Pancreatic progenitor cells, methods and uses related thereto

ABSTRACT

The present invention relates to a substantially pure population of viable pancreatic progenitor cells, and methods for isolating such cells. The present invention further concerns certain therapeutic uses for such progenitor cells, and their progeny.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Applications: U.S.Ser. No. 60/119,576 filed Feb. 10, 1999; U.S. Ser. No. 60/142,305 filedJul. 2, 1999; and U.S. Ser. No. 60/171,338 filed Dec. 21, 1999. Thespecifications of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Insulin-dependent diabetes mellitus (IDDM) is a disease characterized byelevated blood glucose and the absence of the hormone insulin. The causeof the raised sugar levels is insufficient secretion of the hormoneinsulin by the pancreas. In the absence of this hormone, the body'scells are not able to absorb sugar from the blood stream in normalfashion, and the excess sugar accumulates in the blood. Chronicallyelevated blood glucose damages tissues and organs. IDDM is treated withinsulin injections. The size and timing of insulin injections areinfluenced by measurements of blood sugar.

There are over 400 million diabetics in the world today. For instance,diabetes is one of the most prevalent chronic diseases in the UnitedStates, and a leading cause of death. Estimates based on the 1993National Health Interview Survey (NHIS) indicate that diabetes has beendiagnosed in 1% of the U.S. population age <45 years, 6.2% of those age45-64 years, and 10.4% of those age >65 years. In other terms, in 1993an estimated 7.8 million persons in the United States were reported tohave this chronic condition. In addition, based on the annual incidencerates for diabetes, it is estimated that about 625,000 new cases ofdiabetes are diagnosed each year, including 595,000 cases ofnon-insulin-dependent diabetes mellitus (NIDDM) and 30,000 cases ofinsulin-dependent diabetes mellitus (IDDM).

The total cost of diabetes in the United States has been estimated at$92 billion annually, including expenditures on medical products,hospitalization and the value of lost work. Substantial costs to bothsociety and its citizens are incurred not only for direct costs ofmedical care for diabetes, but also for indirect costs, including lostproductivity resulting from diabetes-related morbidity and prematuremortality. Persons with diabetes are at risk for major complications,including diabetic ketoacidosis, end-stage renal disease, diabeticretinopathy and amputation. There are also a host of less directlyrelated conditions, such as hypertension, heart disease, peripheralvascular disease and infections, for which persons with diabetes are atsubstantially increased risk.

While medications such as injectable insulin and oral hypoglycemicsallow diabetics to live longer, diabetes remains the third major killer,after heart disease and cancer. Diabetes is also a very disablingdisease, because medications do not control blood sugar levels wellenough to prevent swinging between high and low blood sugar levels, withresulting damage to the kidneys, eyes, and blood vessels.

Studies have documented that medical costs for persons with diabetes arehigher because they visit physician's offices, hospital outpatientdepartments, and emergency rooms more frequently than their nondiabeticcounterparts, and are more likely to be admitted to the hospital.Americans with diabetes have two to five times higher per capita totalmedical expenditures and per capita out-of-pocket expenses than peoplewithout diabetes. These expenses and their associated loss ofproductivity have impact not only on diabetic patients and theirfamilies, but on federal and state governments and society as a whole.

Data from the Diabetes Control and Complications Trial (DCCT) show thatintensive control of blood glucose significantly delays complications ofdiabetes, such as retinopathy, nephropathy, and neuropathy, comparedwith conventional therapy consisting of one or two insulin injectionsper day. Intensive therapy in the DCCT included multiple injection ofinsulin three or more times per day or continuous subcutaneous insulininfusion (CSII) by external pump. Insulin pumps are one of a variety ofalternative approaches to subcutaneous multiple daily injections (MDI)for approximating physiological replacement of insulin.

Replenishment of functional glucose-sensing, insulin-secretingpancreatic beta cells through islet transplantation has been alongstanding therapeutic target. The limiting factor in this approach isthe availability of an islet source that is safe, reproducible, andabundant. Current methodologies use either cadaverous material orporcine islets as transplant substrates (Korbutt et al., 1997). However,significant problems to overcome are the low availability of donortissue, the variability and low yield of islets obtained viadissociation, and the enzymatic and physical damage that may occur as aresult of the isolation process (reviewed by Secchi et al., 1997;Sutherland et al., 1998). In addition are issues of immune rejection andcurrent concerns with xenotransplantation using porcine islets (reviewedby Weir & Bonner-Weir, 1997).

It is an object of the present invention to create functional beta cellsin vitro by expansion and differentiation of a pancreaticstem/progenitor cell. Advantages lie in obviating the need for physicaldissociation of tissue in order to obtain islets, and the potential forgreater reproducibility and control of the process. Successfulachievement requires the differentiation and maturation ofglucose-sensing, insulin-secreting beta cells from an expandableprecursor population.

SUMMARY OF THE INVENTION

The present invention relates to substantially pure preparations ofviable pancreatic progenitor cells, and methods for isolating such cellsfrom pancreatic ductal tissues, notably intralobular ductal tissue. Thepresent invention further concerns certain uses for such progenitorcells, and their progeny.

In general, the invention features a cellular composition including, asthe cellular component, a substantially pure population of viablepancreatic progenitor cells which progenitor cells are capable ofproliferation in a culture medium. In a preferred embodiment, thecellular composition has fewer than about 20%, more preferably fewerthan about 10%, most preferably fewer than about 5% of lineage committedcells.

In one embodiment, the progenitor cells of the present invention arecharacterized by an ability for self-regeneration in a culture mediumand differentiation to pancreatic lineages. In a preferred embodiment,the progenitor cells are inducible to differentiate into pancreaticislet cells, e.g., β islet cells, α islet cells, δ islet cells, or φislet cells. Such pancreatic progenitor cells may be characterized incertain circumstances by the expression of one or more of: homeodomaintype transcription factors such as STF-1; PAX gene(s) such as PAX6;PTF-1; hXBP-1; HNF genes(s); villin; tyrosine hydroxylase; insulin;glucagon; and/or neuropeptide Y. The pancreatic progenitor cells of thepresent invention may also be characterized by binding to lectin(s), andpreferably to a plant lectin, and more preferably to peanut agglutinin.In certain preferred embodiments, the progenitor cells are PDX1+, e.g.,by FACS sorting, and capable of differentiation into glucose-responsiveinsulin secreting cells.

In certain preferred embodiments, the progenitor cells are PDX1+ andGlut2+. In certain preferred embodiments, the progenitor cells arePDX1⁺, Glut2⁺ and stain with PNA.

In certain preferred embodiments, the subject pancreatic progenitorcells will have one or more of the following characteristics: (i) ableto grow in 2-5 percent fetal calf serum; (ii) able to grow on plastic,e.g., no need to use matrigel; (iii) no statistically significantinduction of cells to proliferate or differentiate when treated withTGFβ5 (GenBank accession P16176) at concenrates up to 30 pg/ml.

In yet another embodiment, the invention features a pharmaceuticalcomposition including as the cellular component, a substantially purepopulation of viable pancreatic progenitor cells, which progenitor cellsare capable of proliferation in a culture medium.

In general, the preferred progenitor cells will be of mammalian origin,e.g., cells isolated from a primate such as a human, from a miniatureswine, or from a transgenic mammal, or are the cell culture progeny ofsuch cells. In one embodiment, pancreatic ductual tissue is isolatedfrom a patient and subjected to the present method in order to provide aresulting culture of pancreatic progenitor cells (or differentiatedcells derived therefrom). Gene replacement or other gene therapy iscarried out ex vivo, and the isolated cells are transplanted back intothe initial donor patient or into a second host patient.

In another aspect, the invention features a cellular compositioncomprising, as a cellular population, at least 75% (though morepreferably at least 80, 90 or 95%) progenitor cells and capable ofself-regeneration in a culture medium.

In yet another aspect, the invention features a cellular compositionconsisting essentially of, as the cellular population, viable pancreaticprogenitor cells capable of self-regeneration in a culture medium anddifferentiation to pancreatic lineages. For instance, in certainembodiments the progenitor cells are isolated from pancreaticintralobular duct explants, e.g. isolated by biopsy, or are the cellculture progeny of such cells.

One aspect of the invention features a method for isolating pancreaticprogenitor cells from a sample of pancreatic duct. In general, themethod provides for a culture system that allows reproducible expansionof pancreatic ductual epithelium while maintaining “stemmedness” and theability to differentiate into endocrine and exocrine cells. Asillustrated below, in a preferred embodiment, pancreatic ductalepithelium is obtained, e.g., by explant or enzymatic digestion, andcultured to confluence. The confluent cell population is contacted withan agent, e.g., a trophic agent such as a growth factor, which causesdifferentiation of progenitor cells in the cultured population.Subsequently, progenitor cells from the explant that proliferate inresponse to the agent are isolated, such as by direct mechanicalseparation of newly emerging buds from the rest of the explant or bydissolution of all or a portion of the explant and subsequent isolationof the progenitor cell population.

In certain embodiments, the culture is contacted with a cAMP elevatingagents, such as8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic-monophosphate (CPT-cAMP)(see, for example, Koike Prog. Neuro-Psychopharmacol. and Biol.Psychiat. 16 95-106 (1992)), CPT-cAMP, forskolin, Na-Butyrate, isobutylmethylxanthine (IBMX) and cholera toxin (see Martin et al. J. Neurobiol.23 1205-1220 (1992)) and 8-bromo-cAMP, dibutyryl-cAMP anddioctanoyl-cAMP (e.g., see Rydel et al. PNAS 85:1257 (1988)).

In certain embodiments, the culture is contacted with a growth factor,e.g., a mitogenic growth factor, e.g., the growth factor is selectedfrom a group consisting of IGF, TGF, FGF, EGF, HGF, hedgehog or VEGF. Inother embodiments, the growth factor is a member of the TGFβsuperfamily, preferably of the DVR (dpp and vg1 related) family, e.g.,BMP2 and/or BMP7.

In certain embodiments, the culture is contacted with a steroid orcorticosteroid such as, for example, hydrocortisone,deoxyhydrocortisone, fludrocortisone, prednisolone, methylprednisolone,prednisone, triamcinolone, dexamethasone, betamethasone andparamethasone. See, generally, The Merck Manual of Diagnosis andTherapy, 15th Ed., pp. 1239-1267 and 2497-2506, Berkow et al., eds.,Rahay, N.J., 1987).

In a preferred embodiment, the cultures are contacted with a cAMPelevating agent, a growth factor and a steroid or corticosteroid, e.g.,with the DCE cocktail described herein.

In another aspect, the invention features, a method for screening acompound for ability to modulate one of growth, proliferation, and/ordifferentiation of progenitor cells obtained by the subject method,including: (i) establishing an isolated population of pancreaticprogenitor cells; (ii) contacting the population of cells with a testcompound; and (iii) detecting one of growth, proliferation, and/ordifferentiation of the progenitor cells in the population, wherein astatistically significant change in the extent of one of growth,proliferation, and/or differentiation in the presence of the testcompound relative to the extent of one of growth, proliferation, and/ordifferentiation in the absence of the test compound indicates theability of the test compound to modulate one of the growth,proliferation, and/or differentiation.

In another aspect, the invention features, a method for treating adisorder characterized by insufficient insulin activity, in a subject,including introducing into the subject a pharmaceutical compositionincluding pancreatic progenitor cells derived by the subject method, ordifferentiated cells arising therefrom, and a pharmaceuticallyacceptable carrier. In preferred embodiments, the progenitor cells arederived from a donor source (which may also be the transplant patient),and expanded at least order of magnitude prior to implantation. As shownin FIG. 40, the subject cellular compositions can be used to rescuediabetic mice.

In a preferred embodiment the subject is a mammal, e.g., a primate, e.g,a human.

In another preferred embodiment the disorder is an insulin dependentdiabetes, e.g, type I diabetes.

In yet another embodiment, the pancreatic progenitor cells are inducedto differentiate into pancreatic islet cells, e.g., β islet cells, αislet cells, δ islet cells, or φ islet cells, subsequent to beingintroduced into the subject.

In other embodiments, the pancreatic progenitors cells are induced todifferentiate into pancreatic islet, e.g., β islet cells, α islet cells,δ islet cells, or φ islet cells, in culture prior to introduction intothe subject.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRITION OF THE DRAWINGS

FIG. 1. Isolation of pancreatic ducts. The cartoon illustrates theprocess by which the ducts under study were obtained. Pancreatic tissuefrom 2-3 week old rats was dissociated in collagenase solution andductal material was obtained by handpicking. Clean ducts were placed inculture on plastic. Within 3-5 days a monolayer was obtained, from whichNACs were produced upon exposure to an inductive medium. Shown below thecartoon is a series of photographs showing the various stages of ductisolation and purity. The first panel shows the primary digest, and thesecond panel shows the result of the first round of handpicking. Notethat there is still some contaminating exocrine tissue. After the secondround of selection, the ducts are free of both islet and exocrinetissue.

FIG. 2. Culture and insulin staining of pancreatic ducts. Depicted is atime series of single ducts in culture from time zero plating (T0) today five (T5) in culture. The upper panel shows examples of Cy3-labelledimmunocytochemical staining for insulin, and the lower panel shows thecombined brightfield and fluorescence images. At T0 there are noinsulin-expressing cells. Within 24 hours of culture, the duct begins todistend and disintegrate, with cells moving toward the periphery.Proliferating cells are present primarily in the outgrowing monolayer ofmesenchymal cells, although ductal epithelial cells do also incorporateBrdU (not shown). Insulin-positive cells emerged spontaneously from theduct over time in culture, but their overall replication rate is slow(not shown). By T5 some monolayers contained sizable clusters ofinsulin-positive cells; typically these contained 20 cells or less.

FIGS. 3A-3G. The duct monolayer expresses multiple progenitor cellmarkers. Monolayers were stained for both insulin (A) and amylase (B).Panel C is a composite showing that some cells express both insulin andamylase. Two morphologically distinct cell types are present, those thatare adherent and flat, and cells that are semi-adherent and round.Arrowheads denote* rounded semi-adherent cells that may coexpress bothinsulin and amylase. Panels D and E show staining for glucagon and PYY,respectively, and Panel F is a composite showing that one of theglucagon-bright cells also expresses PYY. Panel G shows a composite ofnuclear PDX-1 (Cy3) and cytoplasmic insulin (FITC) staining. Arrowheadsindicate cells that express PDX-1 but not insulin or vice versa.

FIGS. 4A-4D. Factor addition influences the appearance of thenonadherent cell (NAC) type. Hoffmnan modulation contrast photographswere taken of cultures treated with various factors. Cultures were grownin 5% FBS for five days until a confluent monolayer was obtained;factors were then added for an additional 48 hours and the cultures werephotographed. NACs were observed in all conditions. Panel A shows thecontrol culture grown in FBS; panel B, culture treated with DCE (1 μMDexamethasone, 100 ng/ml Cholera toxin, 10 ng/ml EGF); Panel C, HGF (10ng/ml), and Panel D, TGFPβ1 (10 ng/ml). Arrow 1 in Panel A indicates theadherent and confluent monolayer and the Arrow 2 points to a pair ofrounded loosely or non-adherent cells. The HGF and TGFβ1 treatedcultures also contained semi- and non-adherent cells. However, thepharmacological cocktail DCE induced, on average, at least 8-fold moreNACs than all other conditions tried. BrdU pulsing experiments showedstrong proliferation and a confluent monolayer even after 48 hours ofDCE exposure, indicating perhaps asymmetric division as opposed tosimple loss of cell adherence. The inset in Panel B illustrates themorphology and granularity of NACs.

FIGS. 5A-5G. Multiple hormone-containing cell types are detected in theNAC population. NACs were collected from DCE-stimulated monolayercultures and analyzed immunocytochemically for endocrine markerexpression. Cells expressing insulin (A, C), PDX-1 (D), glucagon (E),somatostatin (F), and pancreatic polypeptide (G) were all present in theNAC population. Markers were visualized with FITC or Cy3immunofluorescence and the nuclei counterstained with DAPI (C-G). PanelA shows a 10× objective field magnification of insulin staining.Heterogeneous signal strength was observed; shown here are one brightlystained cell and many dimly stained cells along with negative cells.Panel B shows staining with normal preimmune serum. Note that the dimcells in A are significantly above background, yet contain much lessinsulin than the bright cell observed. Panel C shows highermagnification (20× objective) of another insulin staining (Cy3) showingdim and negative cells. In this field and others approximately 40-50% ofthe cells test insulin-positive. Panels D, E, F, and G show PDX-1,glucagon, somatostatin and pancreatic polypeptide staining, respectively(60× objective). Arrows indicate DAPI-stained, hormone-negative cells.

FIGS. 6A-6D. Single cell PCR (SC-PCR) analysis of PDX-1, insulin andglucagon expression in NACs. Forty cells were selected from a randompopulation of NACs and processed for cDNA as described in the Methodssection. These cDNAs were then analyzed for insulin (B), glucagon (C),and PDX-1 (D) message. Panel A shows the ethidium bromide staining ofthe cDNA on a 1.2% agarose gel. The bulk of cDNA product fell within thetargeted 500-1000 bp range. Panel B shows that there is variation in theamount of insulin message per cell, with some cells giving much strongersignal than others. {fraction (15/40)} (37%) of the cells testedpositive for insulin mRNA. Of these, one was also positive for glucagon,and both messages were relatively weak compared to the other cells thatexpressed insulin only. Panel C shows that {fraction (2/40)} (5%) of thecells contained glucagon message, a result that correlates well with theimmunocytochemistry data. Panel D shows that many of the picked cellscontained PDX-1 mRNA. Note that a significant fraction of cells expressPDX-1 mRNA only, with no insulin or glucagon.

FIG. 7. Insulin content and glucose response of cultured ducts. Freshlyisolated ducts (T=0), ducts cultured for one week (T=7), and NACsharvested from the DCE-induced duct culture were all tested forglucose-stimulated insulin secretion (GSIS) and also extracted for totalinsulin content. The time zero ducts contained no detectable insulin byRIA. In contrast, the cultured ducts did have a discernible increase ininsulin content, but no glucose response. In this representativeexperiment, the isolated NACs showed an insulin content that was 18-foldgreater than the DCE-treated monolayer when normalized to a per celllevel. In addition, the NACs demonstrated a strong 3-fold GSIS response,well within the 3-5 fold physiological range observed with adult islets.ML=monolayer, n.d.=not detectable.

FIGS. 8A-8B. Glucose-induced calcium currents in NACs. Glucose inducesan inward calcium current in the NAC population. Changes inintracellular calcium at the single cell level were monitored usingFluo-3 and confocal microscopy. A, In this representative experiment(n>10) approximately one-third of the population initiated a robustcalcium influx in response to glucose administration, and 58% of thecells showed no response to glucose. In every experiment, approximately6-10% of the cells begin with a high intracellular calcium content thatdecreases with time; these were judged to be dying cells. 80 cells wereanalyzed in this experiment. B, The reversibility of the induced calciumcurrent is demonstrated. In this representative experiment (n>6), theglucose-stimulated calcium current could be washed out with Krebs RingerPhosphate (KRP) solution. A second calcium current could then bestimulated by readministration of 17 mM glucose. Washout of the glucosefollowed by tolbutamide stimulation, a SUR-linked potassium channelblocker, also stimulated a calcium current, as expected. Arrows indicatetimes of administration. A total of 123 cells were analyzed in thisexperiment. 7-13% of the cells gave rise to calcium currents in responseto the stimulus (shown in red) whereas 45-65% of the cells showed noresponse to any of the stimuli (shown in blue). The remaining 35% ofcells exhibited varying amplitudes and kinetics in response tochallenge, indicating a complex population.

FIG. 9. Graph showing induction of differentiation by DCE

FIG. 10. Graph showing effect of Forskolin, Dibutyrl cAMP andNa-Butyrate on induction of differentiation.

FIGS. 11 and 12. Graphs illustrating the effect of secretin on inductionof floating progenitor cells.

FIG. 13. Graph demonstrates that Vasoactive Intestinal Peptide (VIP)also differentiates duct monolayers by inducing the appearance offloating progenitor cells.

FIG. 14. Graph showing that insulin diminishes secretin-induceddifferentiation.

FIGS. 15-17. Micrographs that illustate the phenotype of cells whichhave been cultured for two weeks after being sorted on the basis of PNAstaining.

FIGS. 18 and 19. Micrographs that illustrate the specificity of PNA inadult and embryonic pancreas.

FIG. 20. Electrophoretic gel showing the results of typical single cellmRNA PCR amplification reactions.

FIG. 21. Table illustrating the changes in the gene expression duringpancreatic development.

FIG. 22. Chart illustrating one embodiment of an array of markers fordetecting beta cells and precursors thereof.

FIG. 23. Autoradiographs profiling gene expression in adult andembryonic pancreatic tissue, and heart.

FIG. 24. Graph demonstating how quantatitve analysis of gene expressioncan be carried out as part of a determination of the gene expressionprofile of a cell.

FIG. 25. Autoradiographs profiling gene expression in embryonicpancreatic tissue at different stages and after different stimulus.

FIG. 26. Graphs illustating the quantatitve analysis of theautoradiagraphs.

FIG. 27. Autoradiographs profiling gene expression in the so-calledfloating progenitor cells.

FIG. 28. Graph illustating the quantatitve analysis of theautoradiagraphs of FIG. 27.

FIG. 29. Table showing the relative levels of expression of certaingenes between adult islets and during pancreatis development.

FIGS. 30-31. Micrographs that illustrate the binding of certain lectinsto adult rat pancreas.

FIGS. 32-39. Micrographs that illustrate the binding of certain lectinsto adult human pancreas.

FIG. 40. Implanted cells from a pancreatic duct-derived culturetransiently rescues the diabetic state. A heterogeneous populationcontaining functional beta cells derived from the non-adherent portionof a differentiated pancreatic duct monolayer was implanted intostreptozotocin (STZ)-treated diabetic mice. SCID mice injected with STZbecame diabetic within 48 hours. Insulin containing pellets were thenimplanted subcutaneously to stabilize the blood glucose and create amore stable environment for cell implantation. The insulin pellet wasdesigned to expire 7 days post-implantation at T=11 days (T11). Within48 hours of pellet implant the fasting blood glucose of these animalswere reduced from a range of 280-380 mg/dl blood glucose to less than 50mg/dl. In test groups either cells or adult islets as positive controlwere then implanted under the renal capsule. One week later (T13)fasting blood glucose was measured and again at days 16, 21, and 28.Black squares represent placebo group (n=5 mice) and as expected, in theabsence of insulin, the blood glucose slowly climbed over time to wellover 300 mg/dl. Animals (n=5) implanted with insulin pellets only and nocell implants also performed as expected, with a transient rescuefollowed by diabetic rebound after the insulin release tablet hadexpired (red diamonds). Animals receiving islets (blue triangles, n=5,400 islets per animal) showed perfect long term rescue with fastingblood glucose being maintained at approximately 100 mg/dl. The singlesurviving animal receiving duct-derived cells (green circles, n=1 of 7)showed a transient rescue of the diabetic state. The single animaldemonstrated a 4-5 day lowering of >150 mg/dl blood glucose beforerebounding to pre-implant blood glucose levels.

DETAILED DESCRIPTION OF THE INVENTION (i) Overview

The existence within the adult pancreas of a progenitor cell that iscapable of giving rise to the endocrine islet was proposed long ago(e.g., Bensley, 1911). A number of regeneration models provide early invivo evidence for islet neogenesis in the adult organ (Shaw and Latimer,1925, Waren and Root 1925). Histological studies indicated a physicalattachment between what was assumed to be newly forming islets and theductal network. From these and more recent work (Bonner-Weir et al.1993; Gu et al. 1994; Fernandes et al. 1997) developed the now widelyheld belief that islet progenitor cells derive from a subpopulation ofthe pancreatic duct epithelium.

The ductal network is one of three functional components of the adultpancreas (the other two being the exocrine acini and the endocrineislets), and is responsible for fluid secretion and delivery ofdigestive enzymes into the small intestine. Estimates of pancreaticductal mass in the rat average 11%. The exocrine acini, which producethe digestive enzymes, make up by far the greatest portion of the adultpancreas, accounting for approximately 77-89% of overall tissue mass.The islets contain the insulin-secreting beta cells and are responsiblefor hormonal regulation of glucose metabolism; they comprise less than5% of total organ mass (reviewed by Githens, 1988).

Pancreatic regeneration models such as 90% pancreatectomy (Bonner-Weiret al. 1993, Lampeter et al, 1995), duct ligation (Wang et al. 1995,Rosenberg 1995), and transgenic mice (Gu and Sarvetnick, 1993), allprovide further in vivo evidence that islet tissue arises de novo fromduct-associated pancreatic progenitor cells. A common observation ineach of these injury models is the rapid appearance of endocrine cellsin the proliferating ductal epithelium after experimental insult, andthe subsequent appearance over the course of weeks of presumably newlyformed islets in the periductular space. In addition, cells that appearto express both insulin and amylase, which mark endocrine and exocrinecells, respectively, have been observed during the regenerative processand it is thought that these cells might represent activated progenitorcells (Melmed, 1979; Cossel, 1984; Gu et al. 1994). The exact origin ofthese activated cells, whether ductal, acinar, or otherwise, and themechanism of their activation, from progenitor cells or throughselective dedifferentiation, remains to be determined. Despite theseuncertainties, these studies underscore the potential islet neogeniccapacity of the mature pancreas.

It has been proposed that during pancreatic development, earlyexpression of PDX-1, a transcription factor that regulates expression ofinsulin and other beta cell components marks progenitor cells capable ofgiving rise to both the exocrine and endocrine compartments ((Ohlsson etal. 1993; Offield et al. 1996; Ahlgren et al. 1996, and reviewed byMadsen et al. 1996 and Edlund, 1998). Developmental mechanisms may alsoplay a role in adult beta cell neogenesis. Indeed, Fernandes et al.(1997) describe the appearance of PDX-1 expressing cells in adultpancreatic ducts after streptozotocin insult. Another embryonic productis the hormone PYY, whose expression is also postulated to markcommitted endocrine progenitor cells (Upchurch et al. 1994). Both ofthese cell types are found in islets as well as early in pancreaticdevelopment, and it is not yet clear if they contribute directly toislet formation. However they provide early markers against whichregenerative systems can be analyzed.

Long-term in vitro culture of purified adult pancreatic duct epitheliumhas been largely unsuccessful. The work of Githens and his colleaguesrepresents the best attempt to date (Githens et al. 1989; Githens et al.1987). Their approach was to purify the ductal epithelium away fromstromal components during culture, and the results demonstrated that theenriched epithelial fraction could maintain normal epithelial cellsecretory function for a limited time (approximately 1 week) in culture.In addition these cells could express specific pancreatic epithelialmarkers. However, long term cultures were not possible and the authorsdid not report on any observed increase in the formation of endocrinecell types.

Using a different approach, we chose to culture isolated pancreaticducts in their entirety, reasoning that the duct fragment inclusive ofits component epithelium and mesenchyme would be the basic biologicalunit that contains endocrine progenitor cell activity. The dependence ofpancreatic epithelium on its surrounding mesenchyme for survival andgrowth during development was first demonstrated by the early work ofGoloslow and Grobstein (1962) and others (Wessels and Cohen, 1967). Morerecent work using gene knockout technology demonstrated that the loss ofdorsal pancreatic mesenchyme correlated with the loss of the developingpancreatic epithelium (Ahlgren, et al. 1997) and consequently theabsence of pancreas formation from the dorsal bud. Respecification ofpancreatic mesenchyme identity to smooth muscle by sonic hedgehogprotein also resulted in deranged pancreatic epithelial outgrowth(Apelqvist et al. 1997). From these results in the developing pancreas,it was hypothesized that the relationship between mesenchyme andepithelium may continue to be functionally important in the adultpancreas, particularly with respect to islet neogenesis and regrowth.

Freshly isolated duct fragments are comprised of a single epitheliallayer surrounded by mesenchymal stroma and contain few, if any,differentiated endocrine cells. After the ducts are grown in culture, weobserved the presence of multiple endocrine cell types and alsopotential progenitor cells coexpressing markers such as insulin andamylase, which might contribute to the formation of differentiated betacells. In addition, we observed the emergence of a nonadherent cell typepreviously undescribed in pancreatic cell culture. The number andproperties of this novel cell type are affected by the addition ofvarious factors, one combination of which reproducibly leads to theformation of functional, glucose-responsive beta-like cells. Our datathus suggest the presence and induction of pancreatic progenitor cellactivities in this duct culture system, which now makes possible the invitro study of beta cell neogenesis and also provides a first step inthe process of producing beta cells for the treatment ofinsulin-dependent diabetes.

This shows for the first time that functional beta-like cells can beobtained via in vitro duct culture, suggests the presence and activationof a progenitor cell from pancreatic ducts, and provides a system forthe isolation and manipulation of those cells. In one preferredembodiment, the subject method can be used to produce islet-like cellclusters (“ICC”) containing a high percentage of β-epithelial cells withincreased insulin production.

Moreover, as demonstrated in the appended examples, the subject cellularcompositions can be used to rescue diabetic mice.

Accordingly, certain aspects of the present invention relate to isolatedpopulations of progenitor cells capable of subsequent differentiation todistinct pancreatic lineages, methods for isolating such cells andtherapeutic uses for such cells.

In one embodiment, the invention provides a method for isolatingpancreatic progenitor cells. In general, the method includes the stepsof obtaining pancreatic ductal cells; culturing the pancreatic cells ina suitable nutrient medium; isolating a population of progenitor cellsfrom said culture. In preferred embodiment, the ductal epithelial cellsare obtained from intralobular ducts. For instance, the pancreaticductal epithelial cells can be obtained by enzymatic digestion or othermechanical separation of ductal fragments. The pancreatic ductal cellsare grown to confluence, e.g., preferably in a monolayer. Viable,non-adherent cells can be isolated from the culture, optionally aftertreatment of the culture with an agent(s) that induceproliferation/differentiation of pancreatic progenitor cells from theadherent epithelial cells. As described below, the non-adherent cellpopulation is enriched for pancreatic progenitor cells.

Another aspect of the invention relates to cellular compositionsenriched for pancreatic progenitor cells, or the progeny thereof. Incertain embodiments, the cells will be provided as part of apharmaceutical preparation, e.g., a sterile, free of the presence ofunwanted virus, bacteria and other (human) pathogens, as well aspyrogen-free preparation. That is, for human administration, the subjectcell preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biologics standards.

In certain embodiments, such cellular compositions can be used fortransplantation into animals, preferably mammals, and even morepreferably humans. The cells can be autologous, allogeneic or xenogeneicwith respect to the transplantation host. In one aspect, the presentinvention relates to transplantation of fetal or mature pancreatic cellsto treat Type 1 diabetes mellitus.

Another aspect of the present invention relates to our finding that cAMPelevating agents can be used to proliferation and differentiation ofpancreatic progenitor cells. In this regard, the invention relates tothe use of a cAMP elevating agents to induce ex vivo the proliferationand differentiation of pancreatic cells prior to their transplantationinto a diabetic subject. In yet other embodiments, the inventioncontemplates the in vivo administration of cAMP agonists to patientswhich have been transplanted with pancreatic tissue, as well as topatients which have a need for improved pancreatic performance, or areat risk for developing functional deficits in the organ, especially ofglucose-dependent insulin secretion, e.g., the subject method can beused prophylactically.

(ii) Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein the term “animal” refers to mammals, preferably mammalssuch as humans. Likewise, a “patient” or “subject” to be treated by themethod of the invention can mean either a human or non-human animal.

As used herein, the term “cellular composition” refers to a preparationof cells, which preparation may include, in addition to the cells,non-cellular components such as cell culture media, e.g. proteins, aminoacids, nucleic acids, nucleotides, co-enzyme, anti-oxidants, metals andthe like. Furthermore, the cellular composition can have componentswhich do not affect the growth or viability of the cellular component,but which are used to provide the cells in a particular format, e.g., aspolymeric matrix for encapsulation or a pharmaceutical preparation.

The term “culture medium” is recognized in the art, and refers generallyto any substance or preparation used for the cultivation of livingcells. Accordingly, a “tissue culture” refers to the maintenance orgrowth of tissue, e.g., explants of organ primordia or of an adult organin vitro so as to preserve its architecture and function. A “cellculture” refers to a growth of cells in vitro; although the cellsproliferate they do not organize into tissue per se.

Tissue and cell culture preparations of the subject micro-organ explantsand amplified progenitor cell populations can take on a variey offormats. For instance, a “suspension culture” refers to a culture inwhich cells multiply while suspended in a suitable medium. Likewise, a“continuous flow culture” refers to the the cultivation of cells orductal explants in a continuous flow of fresh medium to maintain cellgrowth, e.g. viablity. The term “conditioned media” refers to thesupernatant, e.g. free of the cultured cells/tissue, resulting after aperiod of time in contact with the cultured cells such that the mediahas been altered to include certain paracrine and/or autocrine factorsproduced by the cells and secreted into the culture.

“Differentiation” in the present context means increased numbers ofislet-like cell clusters containing an increased proportion ofbeta-epithelial cells that produce increased amounts of insulin.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a cAMP regulator, with respect to thesubject method, refers to an amount of a cAMP elevating agent which,when added to the subject pancreatic cells cultures, brings about achange in the rate of cell proliferation and/or the state ofdifferentiation of a cell.

The term “explant” refers to a portion of an organ taken from the bodyand grown in an artificial medium.

By “ex vivo” is meant cells that have been taken from a body,temporarily cultured in vitro, and returned to a body.

The term “lineage committed cell” refers to a progenitor cell that is nolonger pluripotent but has been induced to differentiate into a specificcell type, e.g., a pancreatic, cell.

The term “organ” refers to two or more adjacent layers of tissue, whichlayers of tissue maintain some form of cell-cell and/or cell-matrixinteraction to form a microarchitecture.

The term “primary culture” denotes a mixed cell population of humanpancreatic cells that permits interaction of epithelial and mesenchymalcells within ICC. The word “primary” takes its usual meaning in the artof tissue culture.

The term “progenitor cell” refers to an undifferentiated cell which iscapable of proliferation and giving rise to more progenitor cells havingthe ability to generate a large number of mother cells that can in turngive rise to differentiated, or differentiable daughter cells. As usedherein, the term “progenitor cell” is also intended to encompass a cellwhich is sometimes referred to in the art as a “stem cell”. In apreferred embodiment, the term “progenitor cell” refers to a generalizedmother cell whose descendants (progeny) specialize, often in differentdirections, by differentiation, e.g., by acquiring completely individualcharacters, as occurs in progressive diversification of embryonic cellsand tissues. “Progenitor cells” refers to progenitor cells arising intissue of a pancreatic intralobular duct and giving rise to suchdifferentiated progeny as, for example, B cell lineages.

“Proliferation” indicates an increase in cell number.

The term “tissue” refers to a group or layer of similarly specializedcells which together perform certain special functions.

The term “pancreas” is art recognized, and refers generally to a large,elongated, racemose gland situated transversely behind the stomach,between the spleen and duodenum. The pancreatic exocrine function, e.g.,external secretion, provides a source of digestive enzymes. Indeed,“pancreatin” refers to a substance from the pancreas containing enzymes,principally amylase, protease, and lipase, which substance is used as adigestive aid. The exocrine portion is composed of several serous cellssurrounding a lumen. These cells synthesize and secrete digestiveenzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase,ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospholipaseA₂, elastase, and amylase.

The endocrine portion of the pancreas is composed of the islets ofLangerhans. The islets of Langerhans appear as rounded clusters of cellsembedded within the exocrine pancreas. Four different types of cells-α,β, δ, and φ-have been identified in the islets. The a cells constituteabout 20% of the cells found in pancreatic islets and produce thehormone glucagon. Glucagon acts on several tissues to make energyavailable in the intervals between feeding. In the liver, glucagoncauses breakdown of glycogen and promotes gluconeogenesis from aminoacid precursors. The 6 cells produce somatostatin which acts in thepancreas to inhibit glucagon release and to decrease pancreatic exocrinesecretion. The hormone pancreatic polypeptide is produced in the φcells.This hormone inhibits pancreatic exocrine secretion of bicarbonate andenzymes, causes relaxation of the gallbladder, and decreases bilesecretion. The most abundant cell in the islets, constituting 60-80% ofthe cells, is the β cell, which produces insulin. Insulin is known tocause the storage of excess nutrients arising during and shortly afterfeeding. The major target organs for insulin are the liver, muscle, andfat-organs specialized for storage of energy.

The term “pancreatic duct” includes the accessory pancreatic duct,dorsal pancreatic duct, main pancreatic duct and ventral pancreaticduct. Serous glands have extensions of the lumen between adjacentsecretory cells, and these are called intercellular canaliculi. The term“interlobular ducts” refers to intercalated ducts and striated ductsfound within lobules of secretory units in the pancreas. The“intercalated ducts” refers to the first duct segment draining asecretory acinus or tubule. Intercalated ducts often have carbonicanhydrase activity, such that bicarbonate ion may be added to thesecretions at this level. “Striated ducts” are the largest of theintralobular duct components and are capable of modifing the ioniccomposition of secretions.

The term “pancreatic progenitor cell” refers to a cell which candifferentiate into a cell of pancreatic lineage, e.g. a cell which canproduce a hormone or enzyme normally produced by a pancreatic cell. Forinstance, a pancreatic progenitor cell may be caused to differentiate,at least partially, into α, β, δ, or φ islet cell, or a cell of exocrinefate. The pancreatic progenitor cells of the invention can also becultured prior to administration to a subject under conditions whichpromote cell proliferation and differentiation. These conditions includeculturing the cells to allow proliferation and confluence in vitro atwhich time the cells can be made to form pseudo islet-like aggregates orclusters and secrete insulin, glucagon, and somatostatin.

The term “substantially pure”, with respect to progenitor cells, refersto a population of progenitor cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to progenitorcells making up a total cell population. Recast, the term “substantiallypure” refers to a population of progenitor cell of the present inventionthat contain fewer than about 20%, more preferably fewer than about 10%,most preferably fewer than about 5%, of lineage committed cells in theoriginal unamplified and isolated population prior to subsequentculturing and amplification.

(iii) Exemplary Embodiments

Certain terms being set out above, it is noted that one aspect of thepresent invention features a method for isolating pancreatic progenitorcells, and differentiated progeny thereof, from pancreatic tissueexplants, e.g., ductal tissue explants.

One salient feature of the subject method is that the starting materialcan be adult pancreatic tissue. Moreover, the method can be practicedwith relatively small amounts of starting material. Accordingly, smallsamples of pancreatic tissue from a donor can be obtained withoutsacrificing or seriously injuring the donor. The progenitor cells of thepresent invention can be amplified, and subsequently isolated from theepithelial explant, based on a proliferative response upon, for example,addition of defined growth factors or biological extracts to theculture.

Another salient feature of certain embodiments of the subject methodconcerns the use of defined culture conditions for isolating andpropagating discrete pancreatic progenitor cell populations.

For instance, as described below, the progenitor source ductal tissueexplants preferably are digested or otherwise teased apart, therebyproviding purified ductal properties, which in turn are placed in theculture medium and grown. The pancreatic duct preparations are permittedto expand and form monolayer of cells in culture, e.g., a ductalepithelial monolayer. In preferred embodiments, the majority (e.g., >25percent, more preferably >10%) of the cells are vimentin-positive,non-endocrine and proliferative. Viable non-adherent cells (NACs) can beisolated from the culture of otherwise adherent pancreatic cells. Asdescribed below, these NAC preparations are enriched for pancreaticprogenitor cells.

We have also discovered that ductal cells of the subject cultures, andthe NACs arising in the culture, can be purified based on binding tolectins. For example, fluorescently labeled lectins can be used to,e.g., facilitate FACS or other cell sorting. In other embodiments, thelectin can be derivatized for immobilization, e.g., on a solid surfacesuch as a filter or bead, and used for affinity purification of thecells. Exemplary lectins for these purposes include the fluorescentlectins, peroxidase conjugated lectins and biotinylated lectins marketedby Vector Laboratories, Inc. of Burlingame, Calif. In preferredembodiments, the lecting is a plant lectin, and more preferably topeanut agglutinin. In other embodiments, the lectin is selected from thegroup consisting of Aleuria Aurantia Lectin (AAL); Amaranthus CaudatusLectin (ACL, ACA); Bauhinia Purpurea Lectin (BPL, BPA); Concanavalin A(Con A); Succinylated Concanavalin A (Con A); Datura Stramonium Lectin(DSL); Dolichos Biflorus Agglutinin (DBA); Erythrina Cristagalli Lectin(ECL, ECA); Euonymus Europaeus Lectin (EEL); Galanthus Nivalis Lectin(GNL); Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I);Isolectin-B4; Griffonia (Bandeiraea) Simplicifolia Lectin II (GSL II,BSL II); Hippeastrum Hybrid Lectin (HHL, AL); Lens Culinaris Agglutinin(LCA, LcH); Lotus Tetragonolobus Lectin (LTL); Lycopersicon Esculentum(Tomato) Lectin (LEL, TL); Maackia Amurensis Lectin I (MAL I); MaackiaAmurensis Lectin II (MAL II); Maclura Pomifera Lectin (MPL); NarcissusPseudonarcissus Lectin (NPL, NPA, DL); Peanut Agglutinin (PNA);Phaseolus Vulgaris Agglutinin (PHA); Pisum Sativum (PSA); PsophocarpusTetragonolobus Lectin I (PTL I, WBA I); Psophocarpus TetragonolobusLectin II (PTL II, WBA II); Ricinus Communis Agglutinin I (RCA I,RCA120); Ricinus Communis Agglutinin II (RCA II, RCA60, ricin); SambucusNigra (EBL, SNA); Solanum Tuberosum (Potato) Lectin (STL, PL); SophoraJaponica Agglutinin (SJA); Soybean Agglutinin (SBA); Ulex EuropaeusAgglutinin I (UEA I); Ulex Europaeus Aggluutinin II (UEA II); ViciaVillosa Lectin (VVA, VVL); Wheat Germ Agglutinin (WGA); SuccinylatedWheat Germ Agglutinin; and Wisteria Floribunda Lectin (WFA, WFL).

In certain embodiments, the dissociated monolayers derived from theductal explants can be sorted by presence of A2B5 epitope, e.g., theability to be bound by the A2B5 monoclonal antibody (Eisenbarth et al.,(1979) PNAS 76:4913), or other glycolipids present on astrocytes, suchas gangliosides like GM3 or GD3.

Moreover, we have unexpectedly found that a combination of growing suchcells as monolayers, with treatment of such cultures with cAMP elevatingagents produces an increased induction of NACs, e.g., pancreaticprogenitor cells. Accordingly, when the ductal cells have grown toconfluence (cells covering the surface of a culture plate), the cellscans be treated with a cAMP elevating agent in order to causedifferentiation of certain cells in the culture into progenitor cells,and subsequently into insulin-producing or other endocrine or exocrinecells. Accordingly, carefilly defined conditions can be acquired in theculture so as selectively activate discrete populations of cells in thetissue explant. The progenitor and differentiated cells of the presentinvention can be amplified, and subsequently isolated from the culture.

In general, the pancreatic tissue is prepared using any suitable method,such as by gently teasing apart the excised tissue or by digestion ofexcised tissue with collagenase (for example, collagenase A), via, toillustrate, perfusion through a duct or simple incubation of, forexample, teased tissue in a collagenase-containing buffer of suitable pHand tonic strength. The prepared tissue may then, optionally, beconcentrated using suitable methods and materials, such ascentrifugation through ficol gradients for concentration (and partialpurification). The concentrated tissue then is resuspended into anysuitable vessel, such as tissue culture glassware or plasticware. Inpreferred embodiments, the ductal samples are allowed to form aconfluent monolayer culture, from which NACs are formed.

In certain embodiments, the culture is contacted with a cAMP elevatingagents, such as8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic-monophosphate (CPT-cAMP)(see, for example, Koike Prog. Neuro-Psychopharmacol. and Biol.Psychiat. 16 95-106 (1992)), CPT-cAMP, forskolin, Na-Butyrate, isobutylmethylxanthine (IBMX) and cholera toxin (see Martin et al. J. Neurobiol.23 1205-1220 (1992)) and 8-bromo-cAMP, dibutyryl-cAMP anddioctanoyl-cAMP (e.g., see Rydel et al. PNAS 85:1257 (1988)).

In certain embodiments, the culture is contacted with a growth factor,e.g., a mitogenic growth factor, e.g., the growth factor is selectedfrom a group consisting of IGF, TGF, FGF, EGF, HGF, hedgehog or VEGF. Inother embodiments, the growth factor is a member of the TGFβsuperfamily, preferably of the DVR (dpp and vg1 related) family, e.g.,BMP2 and/or BMP7.

In certain embodiments, the culture is contacted with a steroid orcorticosteroid such as, for example, hydrocortisone,deoxyhydrocortisone, fludrocortisone, prednisolone, methylprednisolone,prednisone, triamcinolone, dexamethasone, betamethasone andparamethasone. See, generally, The Merck Manual of Diagnosis andTherapy, 15th Ed., pp. 1239-1267 and 2497-2506, Berkow et al., eds.,Rahay, N.J., 1987).

In a preferred embodiment, the cultures are contacted with a cAMPelevating agent, a growth factor and a steroid or corticosteroid, e.g.,with the DCE cocktail described herein.

A salient feature of the subject method concerns the use of definedexplants as sources from which discrete progenitor cell populations canbe amplified. Thus, in certain embodiments progenitor cells from theexplant that proliferate in response to the agent can be isolated, suchas by direct mechanical separation from the rest of the explant or bydissolution of all or a portion of the explant and subsequent isolationof the progenitor cell population. Moreover, the subject methodgenerally does not require much starting material. Accordingly, smallsamples of pancreatic tissue from a donor can be obtained withoutsacrificing or seriously injuring the donor. The progenitor cells of thepresent invention can be amplified, and subsequently isolated from theepithelial explant, based on a proliferative response upon, for example,addition of defmed growth factors or biological extracts to the culture.

Alternatively, or in addition, treatment with cAMP upregulating agentscan be used as described above to induce differentiation. The cellproducts of such a method can include insulin-producing cells, and morepreferably, glucose-responsive insulin-producing cells.

There are a large number of tissue culture media that exist forculturing tissue from animals. Some of these are complex and some aresimple. While it is expected that the ductal epithelial explants maygrow in complex media, it will generally be preferred that the explantsbe maintained in a simple medium, such as Dulbecco's Minimal EssentialMedia (DMEM), in order to effect more precise control over theactivation of certain progenitor populations in the explant. In apreferred embodiment, the pancreatic ductal epithelium is cultured inIsocave modified MEM cell culture medium with 5% FBS. Moreover, theexplants can be maintained in the absence of sera for extended periodsof time. In preferred embodiments of the invention, the growth factorsor other mitogenic agents are not included in the primary media formaintenance of the cultures in vitro, but are used subsequently to causeproliferation of distinct populations of progenitor cells. See theappended examples.

In general, in such an expanded culture procedure a commercial-sizedbioreactor, such as the OPTICAL TM culture system, Model 5300E (CharlesRiver Labs.; Wilmington, Mass.), or the CELLMAX TM QUAD cell culturesystem (Celico, Inc.; Germantown, Md.), is seeded with a primary cultureof human pancreatic cells. The bioreactor is perfused with a suitable,complete growth medium supplemented with an appropriately effectiveconcentration of mitogens, and as appropriate, cAMP elevating agents.The β-epithelial cell-containing islet-like clusters can then beharvested. Cells may be cryopreserved prior to use as described, forexample, by Beattie et al., Transplantation 56: 1340 (1993).

The cultures may be maintained in any suitable culture vessel, such as a12 or 24 well microplate, and may be maintained under typical cultureconditions for cells isolated from the same animal, e.g., such as 37° C.in 5% CO₂. The cultures may be shaken for improved aeration, the speedof shaking being, for example, 12 rpm.

In order to isolate progenitor cells from the ductal cultures, it willgenerally be desirable to contact the explant with an agent which causesproliferation of one or more populations of progenitor cells in theexplant. For instance, a mitogen, e.g., a substance that induces mitosisand cell transformation, can be used to detect a progenitor cellpopulation in the explant, and where desirable, to cause theamplification of that population. To illustrate, a purified orsemi-purifed preparation of a growth factor can be applied to theculture. Induction of progenitor cells which respond to the appliedgrowth factor can be detected by proliferation of the progenitor cells.However, as described below, amplification of the population need notoccur to a large extent in order to use certain techniques for isolatingthe responsive population.

In yet other embodiments, the ductal explants and/or amplifiedprogenitor cells can be cultured on feeder layers, e.g., layers offeeder cells which secrete inductive factors or polymeric layerscontaining inductive factors. For example, a matrigel layer can be usedto induce hematopoietic progenitor cell expansion, as described in theappended examples. Matrigel (Collaborative Research, Inc., Bedford,Mass.) is a complex mixture of matrix and associated materials derivedas an extract of murine basement membrane proteins, consistingpredominantly of laminin, collagen IV, heparin sulfate proteoglycan, andnidogen and entactin was prepared from the EHS tumor as describedKleinman et al, “Basement Membrane Complexes with Biological Activity”,Biochemistry, Vol. 25 (1986), pages 312-318. Other such matrixes can beprovided, such as Humatrix. Likewise, natural and recombinantlyengineered cells can be provided as feeder layers to the instantcultures.

As described in further detail below, it is contemplated that thesubject methods can be carried out using cyclic AMP (cAMP) agonists toiduce differentiation of the cultures cells of endocrine or exocrinephenotypes. In yet other embodiments, the invention contemplates the invivo administration of cAMP agonists to patients which have beentransplanted with pancreatic tissue, as well as to patients which have aneed for improved pancreatic performance, especially ofglucose-dependent insulin secretion.

In light of the present disclosure, it will be apparent to those in theart that a variety of different small molecules can be readilyidentified, for example, by routine drug screening assays, whichupregulate cAMP-dependent activities. For example, the subject methodcan be carried out using compounds which may activate adenylate cyclaseinclude forskolin (FK), cholera toxin (CT), pertussis toxin (PT),prostaglandins (e.g., PGE-1 and PGE-2), colforsin and β-adrenergicreceptor agonists. β-Adrenergic receptor agonists (sometimes referred toherein as “β-adrenergic agonists”) include albuterol, bambuterol,bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine,dioxethedrine, dopexamine, ephedrine, epinephrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, prenalterol, procaterol, protokylol, reproterol,rimiterol, ritodrine, soterenol, salmeterol, terbutaline, tretoquinol,tulobuterol, and xamoterol.

Compounds which may inhibit cAMP phosphodiesterase(s), and therebyincrease the half-life of cAMP, are also useful in the subject method.Such compounds include amrinone, milrinone, xanthine, methylxanthine,anagrelide, cilostamide, medorinone, indolidan, rolipram,3-isobutyl-1-methylxanthine (IBMX), chelerythrine, cilostazol,glucocorticoids, griseolic acid, etazolate, caffeine, indomethacin,theophylline, papverine, methyl isobutylxanthine (MIX), and fenoxamine.

Certain analogs of cAMP, e.g., which are agonists of cAMP, can also beused Exemplary cAMP analogs which may be useful in the present methodinclude dibutyryl-cAMP (db-cAMP), (8-(4)-chlorophenylthio)-cAMP(cpt-cAMP), 8-[(4-bromo-2,3-dioxobutyl)thio]-cAMP,2-[(4-bromo-2,3-dioxobutyl)thio]-cAMP, 8-bromo-cAMP, dioctanoyl-cAMP,Sp-adenosine 3′:5′-cyclic phosphorothioate, 8-piperidino-cAMP,N⁶-phenyl-cAMP, 8-methylamino-cAMP, 8-(6-aminohexyl)amino-cAMP,2′-deoxy-cAMP, N⁶,2′-O-dibutryl-cAMP, N⁶,2′-O-disuccinyl-cAMP,N⁶-monobutyryl-cAMP, 2′-O-monobutyryl-cAMP,2′-O-monobutryl-8-bromo-cAMP, N⁶-monobutryl-2′-deoxy-cAMP, and2′-O-monosuccinyl -cAMP.

Above-listed compounds useful in the subject methods may be modified toincrease the bioavailability, activity, or other pharmacologicallyrelevant property of the compound. For example, forskolin has theformula:

Modifications of forskolin which have been found to increase thehydrophilic character of forskolin without severly attenuating thedesired biological activity include acylation of the hydroxyls at C6and/or C7 (after removal of the acetyl group) with hydrophilic acylgroups. In compounds wherein C6 is acylated with a hydrophilic acylgroup, C7 may optionally be deacetylated. Suitable hydrophilic acylgroups include groups having the structure —(CO)(CH2)_(n)X, wherein X isOH or NR₂; R is hydrogen, a C₁-C4 alkyl group, or two Rs taken togetherform a ring comprising 3-8 atoms, preferably 5-7 atoms, which mayinclude heteroatoms (e.g., piperazine or morpholine rings); and n is aninteger from 1-6, preferably from 1-4, even more preferably from 1-2.Other suitable hydrophilic acyl groups include hydrophilic amino acidsor derivatives thereof, such as aspartic acid, glutamic acid,asparagine, glutamine, serine, threonine, tyrosine, etc., includingamino acids having a heterocyclic side chain. Forskolin, or othercompounds listed above, modified by other possible hydrophilic acyl sidechains known to those of skill in the art may be readily synthesized andtested for activity in the present method.

Similarly, variants or derivatives of any of the above-listed compoundsmay be effective as cAMP agonists in the subject method. Those skilledin the art will readily be able to synthesize and test such derivativesfor suitable activity.

In certain embodiments, the subject cAMP agonists can be chosen on thebasis of their selectivity for cAMP activation.

In certain embodiments, it may be advantageous to administer two or moreof the above cAMP agonists, preferably of different types. For example,use of an adenylate cyclase agonist in conjunction with a cAMPphosphodiesterase antagonist may have an advantageous or synergisticeffect.

In certain preferred embodiments, the subject agents raise effectivecAMP levels with an ED₅₀ of 1 mM or less, more preferably of 1 μM orless, and even more preferably of 1 nM or less.

In certain embodiments of the subject method, it will be desirable tomonitor the growth state of cells in the culture, e.g., cellproliferation, differentiation and/or cell death. Methods of measuringcell proliferation are well known in the art and most commonly includedetermining DNA synthesis characteristic of cell replication. There arenumerous methods in the art for measuring DNA synthesis, any of whichmay be used according to the invention. In an embodiment of theinvention, DNA synthesis has been determined using a radioactive label(³H-thymidine) or labeled nucleotide analogues (BrdU) for detection byimmunofluorescence.

However, in addition to measuring DNA synthesis, morphological changescan be, and preferably will be, relied on as the basis for isolatingresponsive progenitor cell populations. For instance, as described inthe appended examples, we have observed that certain growth factorscause amplification of progenitor cells in ductal explants so as to formstructures that can be easily detected by the naked eye or microscopy.In an exemplary embodiment, those progenitor cells which respond togrowth factors by proliferation and subsequent formation of outgrowthsfrom the explant, e.g., buds or blebs, can be easily detected. Inanother illustrative embodiment, other structural changes, e.g., changesin optical density of proliferating cells, can be detected via contrastmicroscopy.

To further illustrate, ICCs can be incubated with bromodeoxyuridine(“BrdU”), fixed in formaldehyde, embedded in paraffin and sectioned.Sections can be stained for insulin using an immunoalkaline phosphatasetechnique described, for exampole, by Erber et al., Am. J. Clin. Path.88: 43 (1987), using polyclonal guinea pig anti-porcine insulin(Chemicon; El Sequndo, Calif.) as the primary antibody.

Cell nuclei that have incorporated BrdU during DNA synthesis can beidentified using mouse monoclonal anti-BrdU (Dako; Carpintaria, Calif.),detected with the immuno-peroxide technique of Sternberger et al., J.Histochem., Cytochem. 18: 315 (1970), followed by hematoxylincounterstaining.

Epithelial cells can be identified on separate sections using a mousemonoclonal anti-epithelial antigen antibody (Ber-EP4, Dako, above) asthe primary antibody.

Surface areas of insulin-positive and epithelial cells, calculated aspercent of the total ICC area, can be quantified with a computerizedimage analyzer (American Innovision; San Diego, Calif.). The same methodcan be used for the determination of the BrdU labeling index. Cellspositive for both insulin and BrdU may also be recorded in separatesections of the same samples after double staining of the two antigens.

Mean cell size can be calculated by the ratio of total ICC area to thenumber of nuclei.

Mean beta-cell size can be estimated by measuring the surface area ofindividual insulin-positive cells.

A sufficient number of ICC sections (at least 15) and nuclei (at least1000) should be analyzed for each sample to correct for biological andexperimental variability of the samples.

Various techniques may be employed to isolate the activated progenitorcells of treated explant. Preferred isolation procedures for progenitorcells are the ones that result in as little cell death as possible. Forexample, the activated progenitor cells can be removed from the explantsample by mechanical means, e.g., mechanically sheared off with apipette. In other instances, it will be possible to dissociate theprogenitor cells from the entire explant, or sub-portion thereof, e.g.,by enzymatic digestion of the explant, followed by isolation of theactivated progenitor cell population based on specific cellular markers,e.g., using affinity separation techniques or fluorescence activatedcell sorting (FACS).

To further illustrate, the examples below demonstrate that ductalexplants contain growth factor responsive progenitor cell types. It isfurther demonstrated that different growth factors can induce/amplifydistinct populations of progenitor cells within the ductal tissueexplant to proliferate. This indicates the presence of specific growthfactor receptors on the surface of distinct progenitor cell populations.This is important because the expression of these receptors marks theprogenitor cell populations of interest. Monoclonal antibodies areparticularly useful for identifying markers (surface membrane proteins,e.g., receptors) associated with particular cell lineages and/or stagesof differentiation. Procedures for separation of the subject progenitorcell may include magnetic separation, using antibody coated magneticbeads, affinity chromatography, and “panning” with antibody attached toa solid matrix, e.g., plate, or other convenient technique. Techniquesproviding accurate separation include fluorescence activated cellsorting, which can have varying degrees of sophistication, e.g., aplurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels, etc.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation, biotin, which can beremoved with avidin or streptavidin bound to a support, fluorochromes,which can be used with a fluorescence activated cell sorter, or thelike, to allow for ease of separation of the particular cell type. Anytechnique may be employed which is not unduly detrimental to theviability of the cells.

In an illustrative embodiment, some of the antibodies for growth factorreceptors that exist on the subject progenitor cells are commerciallyavailable (e.g., antibodies for EGF receptors, FGF receptors and/or TGFreceptors), and for other growth factor receptors, antibodies can bemade by methods well known to one skilled in the art. In addition tousing antibodies to isolate progenitor cells of interest, one skilled inthe art can also use the growth factors themselves to label the cells,for example, to permit “panning” processes.

Upon isolation, the progenitor cells of the present invention can befurther characterized in the following manner: responsiveness to growthfactors, specific gene expression, antigenic markers on the surface ofsuch cells, and/or basic morphology.

For example, extent of growth factor responsivity, e.g., theconcentration range of growth factor to which they will respond to, themaximal and minimal responses, and to what other growth factors andconditions to which they might respond, can be used to characterize thesubject progenitor cells.

Furthermore, the isolated progenitor cells can be characterized by theexpression of genes known to mark the developing (i.e., stem orprogenitor) cells for the pancreas.

In an illustrative embodiment, the hepatocyte nuclear factor (HNF)transcription factor family, e.g., HNF1-4, are known to be expressed invarious cell types at various times during pancreas development. Forexample, the progenitor cell may express one or more HNF protein such asHNF1α, HNF2β, HNF2β, HNF3γ, and/or HNF4. The glucose transporter Glut2is also a marker for both early pancreatic cells. Certain of the“forkhead” transcription factors, such as fkh-1 or the like, areunderstood to be markers in early gut tissue.

In another illustrative embodiment, homeodomain type transcriptionfactors such as STF-1 (also known as IPF-1, IDX-1 or PDX) have recentlybeen shown to mark different populations of the developing pancreas.Some LIM genes have also been shown to regulate insulin gene expressionand would also be markers for protodifferentiated β islet cells.Likewise, certain of the PAX genes, such as PAX6, are expressed duringpancreas formation and may be used to characterize certain pancreaticprogenitor cell populations. Other markers of pancreatic progenitorcells include the pancreas specific transcription factor PTF-1, andhXBP-1 and the like. Moreover, certain of the HNF proteins are expressedduring early pancrease development and may used as markers forpancreatic progenitor cells.

Progenitor cells giving rise to pancreatic cells may also express suchas markers as villin and/or tyrosine hydroxylase, as well as secretesuch factors as insulin, glucagon and/or neuropeptide Y.

Other markers which can be scored in the NACs include: Rab3A (Zahraouiet al. (1989) J. Biol. Chem. 12:394; Baldini et al. (1995) PNAS92:4284); vesicle-associated membrane protein 2 (VAMP2, Fujita-Yoshigakiet al. (1996) J. Biol. Chem. 271:13130; and Nielsen et al. (1995) J ClinInvest 96:1834); amylin, and/or A2B5 (Eisenbarth et al., (1979) PNAS76:4913).

In other embodiments, the subject cultures of small ductal epithelialcells, as well as possibly the pancreatic progenitor cells arsingtherefrom, are characterized by binding to lectin(s), and preferably toa plant lectin, and more preferably to peanut agglutinin. In a preferredembodiment, the lectin is peanut agglutinin. In other embodiments, thelectin is selected from the group consisting of Aleuria Aurantia Lectin(AAL); Amaranthus Caudatus Lectin (ACL, ACA); Bauhinia Purpurea Lectin(BPL, BPA); Concanavalin A (Con A); Succinylated Concanavalin A (Con A);Datura Stramonium Lectin (DSL); Dolichos Biflorus Agglutinin (DBA);Erythrina Cristagalli Lectin (ECL, ECA); Euonymus Europaeus Lectin(EEL); Galanthus Nivalis Lectin (GNL); Griffonia (Bandeiraea)Simplicifolia Lectin I (GSL I, BSL I); Isolectin-B4; Griffonia(Bandeiraea) Simplicifolia Lectin II (GSL II, BSL II); HippeastrumHybrid Lectin (HHL, AL); Lens Culinaris Agglutinin (LCA, LcH); LotusTetragonolobus Lectin (LTL); Lycopersicon Esculentum (Tomato) Lectin(LEL, TL); Maackia Amurensis Lectin I (MAL I); Maackia Amurensis LectinII (MAL II); Maclura Pomifera Lectin (MPL); Narcissus PseudonarcissusLectin (NPL, NPA, DL); Peanut Agglutinin (PNA); Phaseolus VulgarisAgglutinin (PHA); Pisum Sativum (PSA); Psophocarpus TetragonolobusLectin I (PTL I, WBA I); Psophocarpus Tetragonolobus Lectin II (PTL II,WBA II); Ricinus Communis Agglutinin I (RCA I, RCA120); Ricinus CommunisAgglutinin II (RCA 11, RCA60, ricin); Sambucus Nigra (EBL, SNA); SolanumTuberosum (Potato) Lectin (STL, PL); Sophora Japonica Agglutinin (SJA);Soybean Agglutinin (SBA); Ulex Europaeus Agglutinin I (UEA I); UlexEuropaeus Aggluutinin II (UEA II); Vicia Villosa Lectin (VVA, VVL);Wheat Germ Agglutinin (WGA); Succinylated Wheat Germ Agglutinin; andWisteria Floribunda Lectin (WFA, WFL).

For instance, as shown the attached figures, various components of humanpancreas can be marked by different lectins. DSL marks inter- andintralobular ducts. LCA appears to mark mesenchyme. ECL marksintralobular ducts without marking larger ducts. Succinylated-Wheat GermAgglutinin marks a subset of main duct cells and is quite restrictedcompared to WGA.

Once isolated and characterized, the subject progenitor cells can becultured under conditions which allow further differentiation intospecific cell lineages. This can be achieved through a paradigm ofinduction that can be developed. For example, the subject progenitorcells can be recombined with the corresponding embryonic tissue to seeif the embryonic tissue can instruct the adult cells to codevelop andcodifferentiate. Alternatively, the progenitor cells can be contactedwith one or more growth or differentiation factors which can inducedifferentiation of the cells. For instance, the cells can be treatedwith an agent such as Forskolin, Di-butyrl cAMP, Na-Butyrate,dexamethasone or cholera toxin, or a growth factor such as TGFβ, such asDVR sub-family member.

In another preferred embodiment, the subject progenitor cells can beimplanted into one of a number of regeneration models used in the art,e.g., a host animal which has undergone partial pancreatectomy orstreptozocin treatment of a host animal.

Accordingly, another aspect of the present invention pertains to theprogeny of the subject progenitor cells, e.g. those cells which havebeen derived from the cells of the initial explant culture. Such progenycan include subsequent generations of progenitor cells, as well aslineage committed cells generated by inducing differentiation of thesubject progenitor cells after their isolation from the explant, e.g.,induced in vitro.

Yet another aspect of the present invention concerns cellularcompositions which include, as a cellular component, substantially purepreparations of the subject progenitor cells, or the progeny thereof.Cellular compositions of the present invention include not onlysubstantially pure populations of the progenitor cells, but can alsoinclude cell culture components, e.g., culture media including aminoacids, metals, coenzyme factors, as well as small populations ofnon-progenitor cells, e.g, some of which may arise by subsequentdifferentiation of isolated progenitor cells of the invention.Furthermore, other non-cellular components include those which renderthe cellular component suitable for support under particularcircumstances, e.g., implantation, e.g., continuous culture.

As common methods of administering the progenitor cells of the presentinvention to subjects, particularly human subjects, which are describedin detail herein, include injection or implantation of the cells intotarget sites in the subjects, the cells of the invention can be insertedinto a delivery device which facilitates introduction by, injection orimplantation, of the cells into the subjects. Such delivery devicesinclude tubes, e.g., catheters, for injecting cells and fluids into thebody of a recipient subject. In a preferred embodiment, the tubesadditionally have a needle, e.g., a syringe, through which the cells ofthe invention can be introduced into the subject at a desired location.The progenitor cells of the invention can be inserted into such adelivery device, e.g., a syringe, in different forms. For example, thecells can be suspended in a solution or embedded in a support matrixwhen contained in such a delivery device. As used herein, the term“solution” includes a pharmaceutically acceptable carrier or diluent inwhich the cells of the invention remain viable. Pharmaceuticallyacceptable carriers and diluents include saline, aqueous buffersolutions, solvents and/or dispersion media. The use of such carriersand diluents is well known in the art. The solution is preferablysterile and fluid to the extent that easy syringability exists.Preferably, the solution is stable under the conditions of manufactureand storage and preserved against the contaminating action ofmicroorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. Solutions of the invention can be prepared by incorporatingprogenitor cells as described herein in a pharmaceutically acceptablecarrier or diluent and, as required, other ingredients enumerated above,followed by filtered sterilization.

Support matrices in which the progenitor cells can be incorporated orembedded include matrices which are recipient-compatible and whichdegrade into products which are not harmful to the recipient. Naturaland/or synthetic biodegradable matrices are examples of such matrices.Natural biodegradable matrices include plasma clots, e.g., derived froma mammal, and collagen matrices. Synthetic biodegradable matricesinclude synthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Other examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are known in theart. See e.g., U.S. Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701.These matrices provide support and protection for the fragile progenitorcells in vivo and are, therefore, the preferred form in which theprogenitor cells are introduced into the recipient subjects.

The present invention also provides substantially pure progenitor cellswhich can be used therapeutically for treatment of various disordersassociated with insufficient functioning of the pancreas.

To illustrate, the subject progenitor cells can be used in the treatmentor prophylaxis of a variety of pancreatic disorders, both exocrine andendocrine. For instance, the progenitor cells can be used to producepopulations of differentiated pancreatic cells for repair subsequent topartial pancreatectomy, e.g., excision of a portion of the pancreas.Likewise, such cell populations can be used to regenerate or replacepancreatic tissue loss due to, pancreatolysis, e.g., destruction ofpancreatic tissue, such as pancreatitis, e.g., a condition due toautolysis of pancreatic tissue caused by escape of enzymes into thesubstance.

In an exemplary embodiment, the subject progenitor cells can be providedfor patients suffering from any insulin-deficiency disorder. Forinstance, each year, over 728,000 new cases of diabetes are diagnosedand 150,000 Americans die from the disease and its complications; thetotal yearly cost in the United States is over 20 billion dollars(Langer et al. (1993) Science 260:920-926). Diabetes is characterized bypancreatic islet destruction or dysfunction leading to loss of glucosecontrol. Diabetes mellitus is a metabolic disorder defined by thepresence of chronically elevated levels of blood glucose(hyperglycemia). Insulin-dependent (Type 1) diabetes mellitus (“IDDM”)results from an autoimmune-mediated destruction of the pancreaticβ-cells with consequent loss of insulin production, which results inhyperglycemia. Type 1 diabetics require insulin replacement therapy toensure survival. Non-insulin-dependent (Type 2) diabetes mellitus(“NIDDM”) is initially characterized by hyperglycemia in the presence ofhigher-than-normal levels of plasma insulin (hyperinsulinemia). In Type2 diabetes, tissue processes which control carbohydrate metabolism arebelieved to have decreased sensitivity to insulin. Progression of theType 2 diabetic state is associated with increasing concentrations ofblood glucose, and coupled with a relative decrease in the rate ofglucose-induced insulin secretion.

The primary aim of treatment in both forms of diabetes mellitus is thesame, namely, the reduction of blood glucose levels to as near normal aspossible. Treatment of Type 1 diabetes involves administration ofreplacement doses of insulin. In contrast, treatment of Type 2 diabetesfrequently does not require administration of insulin. For example,initial therapy of Type 2 diabetes may be based on diet and lifestylechanges augmented by therapy with oral hypoglycemic agents such assulfonylurea. Insulin therapy may be required, however, especially inthe later stages of the disease, to produce control of hyperglycemia inan attempt to minimize complications of the disease, which may arisefrom islet exhaustion.

More recently, tissue-engineering approaches to treatment have focusedon transplanting healthy pancreatic islets, usually encapsulated in amembrane to avoid immune rejection. Three general approaches have beentested in animal models. In the first, a tubular membrane is coiled in ahousing that contained islets. The membrane is connected to a polymergraph that in turn connects the device to blood vessels. By manipulationof the membrane permeability, so as to allow free diffusion of glucoseand insulin back and forth through the membrane, yet block passage ofantibodies and lymphocytes, normoglycemia was maintained inpancreatectomized animals treated with this device (Sullivan et al.(1991) Science 252:718).

In a second approach, hollow fibers containing islet cells wereimmobilized in the polysaccharide alginate. When the device was placeintraperitoneally in diabetic animals, blood glucose levels were loweredand good tissue compatibility was observed (Lacey et al. (1991) Science254:1782).

Finally, islets have been placed in microcapsules composed of alginateor polyacrylates. In some cases, animals treated with thesemicrocapsules maintained normoglycemia for over two years (Lim et al.(1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Acta.840:133; Sugamori et al. (1989) Trans. Am. Soc. Artif Intern. Organs35:791; Levesque et al. (1992) Endocrinology 130:644; and Lim et al.(1992) Transplantation 53:1180). However, all of these transplantationstrategies require a large, reliable source of donor islets.

The pancreatic progenitor cells of the invention can be used fortreatment of diabetes because they have the ability to differentiateinto cells of pancreatic lineage, e.g., β islet cells. The progenitorcells of the invention can be cultured in vitro under conditions whichcan further induce these cells to differentiate into mature pancreaticcells, or they can undergo differentiation in vivo once introduced intoa subject. Many methods for encapsulating cells are known in the art.For example, a source of β islet cells producing insulin is encapsulatedin implantable hollow fibers. Such fibers can be pre-spun andsubsequently loaded with the β islet cells (Aebischer et al. U.S. Pat.No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al.(1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res.82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), orcan be co-extruded with a polymer which acts to form a polymeric coatabout the β islet cells (Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat.No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; andAebischer et al. (1991) Biomaterials 12:50-55).

Moreover, in addition to providing a source of implantable cells, eitherin the form of the progenitor cell population of the differentiatedprogeny thereof, the subject cells can be used to produce cultures ofpancreatic cells for production and purification of secreted factors.For instance, cultured cells can be provided as a source of insulin.Likewise, exocrine cultures can be provided as a source for pancreatin.

Yet another aspect of the present invention provides methods forscreening various compounds for their ability to modulate growth,proliferation or differentiation of distinct progenitor cell populationsfrom pancreatic ductal epithelial culture. In an illustrativeembodiment, the subject progenitor cells, and their progeny, can be usedto screen various compounds or natural products. Such explants can bemaintained in minimal culture media for extended periods of time (e.g.,for 7-21 days or longer) and can be contacted with any compound, e.g.,small molecule or natural product, e.g., growth factor, to determine theeffect of such compound on one of cellular growth, proliferation ordifferentiation of progenitor cells in the explant. Detection andquantification of growth, proliferation or differentiation of thesecells in response to a given compound provides a means for determiningthe compound's efficacy at inducing one of the growth, proliferation ordifferentiation in a given ductal explant. Methods of measuring cellproliferation are well known in the art and most commonly includedetermining DNA synthesis characteristic of cell replication. There arenumerous methods in the art for measuring DNA synthesis, any of whichmay be used according to the invention. In an embodiment of theinvention, DNA synthesis has been determined using a radioactive label(³H-thymidine) or labeled nucleotide analogues (BrdU) for detection byimmunofluorescence. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the compound. A control assay can also be performed toprovide a baseline for comparison. Identification of the progenitor cellpopulation(s) amplified in response to a given test agent can be carriedout according to such phenotyping as described above.

(iv) Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE 1 Isolation of Pancreatic Progenitor Cells

METHODS

Duct Isolation and Culture

Pancreata from two litters of 2 week old Sprague-Dawley rat pups wereisolated and placed into 10 ml of (1 U/ml in DMEM) Collagenase A(Boehringer-Mannheim, St. Louis) and digested for 40 min at 37° C. in ashaking water bath at 150-175 rpm. The digest was vortexed briefly andwashed once with C⁺⁺/Mg⁺⁺-free HBSS (Gibeo BRL, Grand Island, N.Y.). Thepellet was resuspended in HBSS and filtered through a 500 μm mesh(Costar Corning, Cambridge, Mass.) and washed again. The pellet wasresuspended to 50 ml in HBSS; 10 ml was transferred to a 10 cm cultureplate (Costa & Corning) and placed under a dissecting scope. Individualduct fragments were selected by aspiration with a micropipette andtransferred to a plate containing medium with serum. The fragments wereselected from this plate a second time, transferred to a fresh tube ofmedium and serum, and washed before being plated. Fragments werecultured on plastic in Iscoveis modified DMEM (Gibco-BRL) containing 5%FBS (Gibco-BRL), glutamine and 1% Pen/Strep (Gibco-BRL). For the studyof individual duct fragments, ducts were grown on 8 well chamber slides(Lab-Tek, Naperville Ill.). For generating monolayers or NACs, ductfragments were plated to four 4-well plates (Nunc) per prep. Inductionof NACs was achieved by a medium change after confluence (generally day5 in culture) to Iscoveis DMEM supplemented with 5% FBS, glutamine,Pen/Strep and Dexamethasone (1 μM, Sigma), Cholera Toxin (100 ng/ml,Sigma), and EGF (10 ng/ml, Gibco). NACs were harvested after 48 hours.

Immunocytochemisty

Cultures, ducts, and non-adherent cells were fixed in 1%paraformaldehyde and permeabilized in PBS containing 0.3% TritonX-100(PBST). Nonspecific binding sites were blocked by preincubation in ablocking buffer consisting of 5% normal donkey serum (JacksonImmunoResearch) and 1% BSA (Sigma) in PBST. All antibodies were dilutedin this blocking buffer. Incubations with primary antibody were carriedout overnight at 4° C. in a humidified chamber. Primary antibodies usedwere: guinea pig anti-insulin (Linco, 1:2000); mouseanti-insulin/proinsulin (Biodesign, directly conjugated to biotin usinga labeling kit from Boehringer Mannheim); guinea pig anti-glucagon(Linco, 1:2500), mouse anti-somatostatin (Biomeda, 1:50); rabbitanti-pancreatic polypeptide (Zymed, 1:50); rabbit anti-amylase (Sigma,1: 1500), and rabbit anti-PDX-1 (gift of Christopher Wright, Vanderbilt,1:2000). Secondary antibodies and tertiary reagents were:FITC-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch,1:200); Cy3-conjugated donkey anti-guinea pig, rabbit, or mouse IgG(Jackson, 1:1000); biotin-conjugated donkey anti-rabbit or mouse IgG(Jackson, 1:500); AvidinD-FITC (Vector Labs, 1:1000); streptavidin-Cy3(Jackson, 1:1000). Cells were scored on a Nikon Eclipse E800epifluorescent or Nikon Diaphot 300 inverted fluorescent/phasephotomicroscope.

Single Cell cDNA Amplification and PCR Analysis

cDNAs from single cells were amplified according to Brady et al. (1993)and Dulac and Axel (1995). Single NACs were randomly picked andtransferred into PCR tubes containing ice-cold lysis buffer. The firststrand cDNA synthesis and subsequent PCR amplification were performedexactly as described (Dulac and Axel, 1995) except that the PCRreactions were performed in a total volume of 50 μl instead of 100 μl.The amplified cDNAs were electrophoresed on a 1% agarose gel and thesize of DNA fragments ranged from 0.5-1 kb as expected. The aliquots ofindividual cDNAs were then analyzed for marker genes by PCR usingspecific PCR primers. The PCR reactions were run for 35 cycles each at94° C. for 30 sec, 55° C. for 1 min, and 72° C. for 2 min. Amplimersequences were: ATGTCGTCCAGGCCGCTCTGGACAAAATATGAATTCT₂₄ (SEQ ID NO.1);insulin: 5{acute over (1)} primer, CACAACTGGAGCTGGGTGGAG (SEQ ID NO.2);3{acute over (1)} primer, CAAAGGCTTTATTCATTGCAGAGG (SEQ ID NO.3); PDX-1:5{acute over (1)} primer, GACCGCAGGCTGAGGGTGAG (SEQ ID NO.4); 3{acuteover (1)} primer, CAGAGGTCTGCCAGCATCTCG (SEQ ID NO.5); glucagon: 5{acuteover (1)} primer, TCCCAGAAGAAGTCGCCATTG (SEQ ID NO.6); 3{acute over (1)}primer, TTCATTCCGCAGAGATGTTGRG (SEQ ID NO.7); beta-actin: 5′ primer, AAGTCC CTC ACC CTC CCA AAA G (SEQ ID NO.8); 3′ primer, AAC ACC TCA AAC CACTCC CAG G (SEQ ID NO.9).

Insulin Release Assay

Insulin release was measured under static incubation conditions usingNACs, isolated ducts, monolayer cells, or batches of 10 islets. Cells orislets were preincubated in Krebs Ringer Phosphate buffer (KRP)containing 3 mM Glucose (Sigma) and 0.2% BSA (Sigma) for 30 minutes at37° C. Supernatant was collected and the cells washed once beforefurther incubation in 17 mM glucose for 1 hour at 37° C. Thissupernatant was then collected and all samples were kept at −20° C.until the insulin-specific radioinununoassay was performed using a RIAkit for rat C-peptide from Linco Research (St. Charles, Mo.). Forinsulin content measurements, cells were extracted in acid-ethanol andsonicated prior to assay.

Calcium Imaging

NACs were immobilized in 0.7% low melting agarose in Hanks buffer(GIBCO) and dye-loaded for 1 hour at room temperature with 5 μM Fluo-3acetoxy-methyl (AM) ester (Molecular Probes) in standard Krebs RingerPhosphate (KRP) buffer that additionally contained 0.1% pluronic acid(Molecular Probes) and 1% dimethyl sulfoxide (Sigma). Cells were thenwashed to remove excess dye and placed onto a heated microscope stage 30(Olympus) and maintained at 32° C. Fluo-3 fluorescence intensity wasused as an indicator of intracellular calcium concentration and wasmeasured with a confocal laser-scanning microscope (Olympus). Theexcitation wavelength was set to 488 nm (argon ion laser) and a 40×water lens was used. The same parameters for laser scanning were set foreach experiment, including confocal aperture and laser intensity. Thelaser scanning was performed as an XYT series with an interval of 10seconds between each scan in order to resolve glucose-induced changes inintracellular calcium. The imaging files were stored and subsequentlyanalyzed with FLUOVIEW software (Olympus). Cells of interest werecircled and the mean intensity of the circled areas was plotted overtime.

RESULTS

Isolation, Characterization and Culture of Pancreatic Ducts

In order to establish a defined in vitro culture system, a population ofinterlobular ducts was isolated and characterized. Adult tissue from 1-2month old animals was initially used to establish the described culturesystem, but was eventually replaced with tissue from 2-3 week old ratswhich provide a more consistent and greater yield of clean ducts.Pancreatic tissue was harvested and subjected to collagenase digestion(Githens & Whelan, 1983; Githens et al. 1989). Digested tissue was thenhandpicked in multiple iterations until a pure population of ducts wasobtained (FIG. 1). A typical experiment provided up to 200-300 fairlyuniform duct fragments per 20 animals. Staining for tubulin beta III andacetylated-LDL-DiI showed that the selected duct population was free ofneurons and blood vessels (not shown).

Characterization of the starting material was performed by analyzingsingle ducts for expression of insulin, PDX-1, PYY, and amylase. Table 1shows that the majority of the handpicked ducts at time zero were freeof these endocrine and exocrine markers. Greater than 92% of all ductscontained no insulin-immunopositive cells at the start of culture, and asimilar proportion of all ducts had no immunohistochemically detectablePDX-1. Of those ducts that tested positive, almost all had only 1-2cells that were insulin immunopositive. Analysis of dissociated ductsalso showed that less than 0.05% of time zero duct cells wereimmunopositive for insulin and PDX-1 protein (not shown). Similarly,PYY-containing cells were rare, constituting no more than 0.015% of thecounted cells. Amylase-positive cells constituted 0.02% of the initialpopulation and probably represented exocrine carryover since theyoccurred in rare clusters. The number of cells expressing either PDX-1or the endocrine markers insulin or PYY totaled much less than 0.1% ofthe cells (Table 1), corroborating the immunohistochemical observationson sections of adult pancreas that these cells are rare in mature ducts(data not shown). Since the average duct fragment contained 3450±1860cells (n=10 determinations), and the average duct yield was 225fragments, the initial number of insulin positive cells at the start ofculture ranged from 80-400 per approximately 800,000 cells (Table 1).

Culturing was performed by placing single duct fragments within a 1 cm²well (FIG. 2) or multiple fragments into 4-well plates (1.9 cm²/well).Various substrates were tested (Matrigel, collagen, hydrogels) but thecleanest and most interesting results were obtained by simply platingonto charged plastic. Iscoveis Modified Dulbeccois Media (IMDM)containing 5% fetal calf serum (FCS) was added to each well and theducts cultured over 5 days. The top panel in FIG. 2 shows insulinstaining in a time series of cultured ducts and the bottom panel thecorresponding bright field images. An analysis of single ductpopulations indicated that whereas at time zero 8% of the duct fragmentswere positive for insulin (Table 1), in as short a time as 24 hours, thenumber of ducts containing insulin-positive cells had increased to 13%and to 17% by day two. These positives appeared most frequently assingle cells or small foci of 2-4 cells (FIG. 2). By day five ofindividual culture 23-25% of the wells contained insulin-positive cellson the monolayer, with little change thereafter (through day seven,{fraction (46/185)} single duct cultures). One implication of theseresults is that non-insulin positive ducts become insulin-positivethrough culture.

Increasing the number of duct fragments per well resulted in a morerapid outgrowth and confluence of the monolayer generally within 5 days,suggesting that some cross-feeding takes place. We have standardized ourplatings to 16 wells (1.9 cm²) per duct preparation as a balance betweentime and cell yield. At seven days of culture in FBS(T7), the monolayercontains an average of 25±20 (range 0-51) insulin-positive cells perwell (0.02% of total cells) for a maximum expansion of 5-fold over thestarting material. In this system, the majority of cells arevimentin-positive, non-endocrine and proliferative (as indicated by BrdUuptake), and probably arise from the stromal cells surrounding theepithelial layer of duct cells. The insulin-positive cells are capableof BrdU uptake but this occurs rarely (not shown). The bulk of BrdUuptake is by vimentin-positive fibroblasts. The slow growth rate of theearly appearing insulin-positive cells on the monolayer agrees withobservations from other investigators demonstrating that beta cellsreplicate very infrequently in vitro and in vivo (reviewed by Sjoholm,1996; Nielsen et al. 1999). Further culture beyond seven days did notsignificantly increase either the number of wells containinginsulin-positive cells or the number of insulin-positive cells.

In addition to an increase in the number of cells expressing insulin, wealso observed that some wells in FBS culture contained a number ofamylase-positive cells, often cells that coexpressed insulin (FIGS.3A-C). Cells expressing both insulin and amylase have been documented toappear during pancreatic regeneration and are thought to be activatedprogenitor cells (Melmed, 1979, Ou et al. 1994). In addition, thesecultures contained a rounded and semi-adherent cell type, many of whichappear to express both markers although the insulin is faint.

Cells that express PYY and/or glucagon are observed in our duct cultures(FIG. 3D-F). The coexpression of PYY and glucagon during earlypancreatic development has been postulated to mark endocrine progenitorcell progression (Upchurch et al. 1994). In contrast to insulin, thenumber of PYY-positive cells did not change during culture (Table 2). Ofcells that were insulin-positive on the monolayer, most but not allcoexpressed PDX-1. Cells that expressed PDX-1 but did not yet expressinsulin and vice versa were also observed in culture (FIG. 3G, arrows).Thus cells representing various stages of differentiation and differentdevelopmental lineages appear in our cultures.

Appearance of a Non-adherent Cell Type

The number of insulin-positive cells did not increase significantly withculture beyond 5 days due to their slow replication rate and perhapscell death. Factors were added to T5 cultures to determine if anincrease in the number of insulin-positive cells could be induced. Anumber of factors that affect either epithelial cells or pancreaticdevelopment were tested: dexamethasone, cholera toxin, EGF, TGFα, PDGFα,HGF, TGFβ1, IL-1α, GLP-1, glucagon, gastrin, GIP, PYY, NPY, and PP wereadded to T5 cultures for 2 further days of incubation. When tested alonemost of the factors did not significantly boost the number of observedinsulin-positive cells (not shown). However, a cocktail of DCE,dexamethasone, cholera toxin and EGF, significantly increased the numberof insulin-positive cells on the monolayer (an average increase of 2-3fold over n=8 experiments, Table 1). In addition, the presence of DCEsignificantly enhanced the appearance of a population of non-adherentcells (NACs) over the course of 48 hours (FIG. 4). NACs were observedeven in control cultures (FIG. 4A) as well as in growth factor-treatedcultures (FIGS. 4C,D), but none of these conditions led to the level ofinduction seen with DCE. In the examples shown, HGF and TGFβ1 weretested for effects on the monolayer. HGF has been shown to stimulate thegrowth of fetal islets (Otonkoski et al. 1994) and TGFβ1 has been shownto inhibit the appearance of endocrine cells in in vitro pancreaticculture (Sanvito et al. 1994). In our system, HGF and TGFβ1 had onlyslight effects on culture phenotype or NAC production.

NACs appear spontaneously in confluent monolayer cultures. Both thenumber and rate of appearance of NACs are significantly increased(often >8-fold) by the addition of the DCE cocktail (FIG. 4B). Thesecells are characteristically phase-bright, possess a secretoryappearance with high granularity, and generally range in size from 20-50μm (FIG. 4 inset). True NACs most often appear as large round cellsfreely floating at the surface of the monolayer. Many others are oftenstill attached and apparently in the process of emerging. An increase inNACs can be seen by 24 hours post-DCE addition, but appears maximal at48 hours. Repeated dosing of DCE into the cultures gave rise tosuccessive waves of NAC formation but with successively fewer numbers(not shown).

DCE has previously been shown to promote the growth and function ofprimary purified pancreatic epithelial cultures (Githens et al. 1987,1989), but no NACS or endocrine cell types were reported. Perhaps theeffect of DCE is indirect; working on the stromal component of our mixedcell culture system to induce the differentiation of beta and otherislet cell types. Subsequent testing in our cultures showed that neitherdexamethasone not EGF alone had significant effects on NAC generation incomparison to controls, but that the majority of the activity wasassociated with the cAMP-elevating effects of the cholera toxin. Infact, many cAMP agonists also had this effect (data not shown; to bedescribed elsewhere). The presence of dexamethasone and EGF appeared toenhance the effect of CT. The size and granularity of the cells withinthe NAC population varied markedly, but vital dye staining showedgreater than 99% of the NACs to be viable.

In terms of the number of ducts that were responsive to DCE, over 95% ofthe DCE-treated wells (n>10 experiments, single duct per well) gave riseto at least a 2-fold increase in NACs over control wells. Each treatedwell in a normal culture (8-16 ducts per well) yielded 3,000-18,000 NACsafter 48 hours in DCE (n=9 experiments) with an average yield of about7000 per well or approximately 1×10⁵ NAC cells per prep. BrdUincorporation experiments demonstrated that one of the effects of DCE isto stimulate cell division. Pulsing at the end of the 48 hours showed4-fold more BrdU-positive cells in the DCE-treated monolayers than incontrols, indicating a long-lasting stimulation of proliferation (notshown). When pulsed at the beginning of DCE addition, 10% of the NACsrecovered at 48 hours were BrdU-positive, indicating that these cellsmay derive from a DCE-responsive, cycling cell. DCE does not appearsimply to stimulate the loss of cell adhesion.

Hormone Expression in NACs

Analysis of the monolayer demonstrated that approximately 0.02% of thecells in FBS culture expressed insulin (see Table 1), with DCE additionincreasing that number 2-3 fold to give an average of 59±52 (range5-196) insulin-positive cells per well. In addition to analysis of themonolayer, NACs were analyzed for expression of insulin and otherendocrine markers. Since NACs are free-floating in the media, they werecollected by aspiration at 48 hours post DCE addition, when theirappearance was maximal. All four islet endocrine cell types could bedetected immunocytochemically in this population. FIG. 5 shows NACimmunostaining for insulin, PDX-1, glucagon, somatostatin, andpancreatic polypeptide. As shown in FIG. 5A, insulin labelingdemonstrated a continuum of fluorescence intensity with approximately4-5% of the cells consistently bright (range 2.5-13%, n=6 measurements),and the majority of positive cells (>30-40% of the total cellpopulation) exhibiting low levels of immunofluorescence (A, C) that werestill above background (B). This number of low-insulin-expressing cellswas confirmed by FACS analysis (data to be described elsewhere). Cellsexpressing PDX-1, glucagon, somatostatin, and pancreatic polypeptidewere also present in the population (D, E, F and G respectively), withglucagon-positive cells being the next most frequent (6%), followed bysomatostatin (3%) and pancreatic polypeptide cells the rarest (2%). TheNAC population thus constitutes an enrichment from a cultured monolayerof the full set of islet endocrine cell types.

Transcriptional Profiling

As a further determination of the number of insulin-expressing cellswithin the NAC population, we also performed semi-quantitativesingle-cell PCR (Brady et al. 1993, Dulac & Axel, 1995) to detectinsulin mRNA in randomly selected individual NACs. FIG. 6 shows that 15of 40 or >35% of the cells analyzed contained insulin mRNA. Panel Ademonstrates that cDNA was amplified from each single cell sample. PanelB shows that the insulin message varied in intensity among the positivecells. This variation in signal intensity, similar to that observed byimmunocytochemisty, was also observed and verified by arrayhybridization analysis (data not shown). Two of the 40 selected cellscontained glucagon message (Panel C) with one of them also containinginsulin and PDX-1 message. Panel D shows that greater than 80%({fraction (35/40)}) of the cells analyzed contained PDX-1 message. Onlyone of the insulin-positive cells did not express PDX1, whereas therewere many cells that were PDX-1 positive with no detectable insulin orglucagon message. The absolute and relative numbers of insulin andglucagon cells are in good agreement with that observed byimmunocytochemistry, and in the case of insulin, with flow analysis aswell. Interestingly, there were a number of NACs that did not expressany of the three markers. The identities of these cells is currentlyunknown. Array hybridization of the 40 cDNAs with a labeled probe to theribosomal component S6 (RPS6) demonstrated its presence in all samples(not shown). Transcriptional profiling confirms the immunocytochemistryresults that many of the NACs express PDX-1, that approximately 40% ofthe cells are both insulin mRNA and protein positive, albeit at varyinglevels, and that the majority of NACs appear to have a beta cellphenotype. In addition, there is a significant fraction of cells thatare PDX-1 positive but insulin-negative, which might indicate progenitorcell status. SC-PCR of monolayer cells showed 23/23 actin-positive, 0/23insulin positive cells (not shown), demonstrating the relativeenrichment of endocrine phenotypes in the NAC population.

Insulin Content and Glucose-stimulated Insulin Secretion

A hallmark of functional beta cells is their ability to secrete insulinin response to elevated glucose levels. In order to determine whetherthere are functional beta cells within the duct cultures, we performedstatic insulin release assays on both the monolayer and the NACpopulations to determine their responses to glucose challenge. We alsomeasured total insulin content by RIA in order to determine the relativeincrease in insulin expression due culture. FIG. 7 shows theglucose-induced insulin release in isolated time zero ducts,DCE-cultured monolayer, and harvested NACs. The NAC populationdemonstrated a 3-fold increase in secreted insulin in response toelevated glucose. In contrast, the cells contained in the monolayerexhibited little glucose response and secreted far less insulin ineither high or low glucose conditions. Analysis of the time zero ducts(n=3) showed no glucose-stimulated insulin secretion and acid-ethanolextraction of the ducts showed no detectable insulin within the level ofsensitivity (approx. 100 μg/ml) of the RIA. Extraction for total insulinshowed a content of 1.34 ng of insulin per 50,000 cells on themonolayer, and 25.0 ng of insulin per 50,000 NAC cells. In comparison, anormal rat islet of approximately 1000 cells typically contains about 20ng of insulin (data not shown). Furthermore, when NACs recovered fromcultures exposed to DCE for 24, 48, or 72 hours were compared, only theNACs from the 48-hour cultures demonstrated a reliableglucose-stimulated insulin response. The data thus show a large increasein the amount of insulin produced through duct culture and that theinsulin can be productively released in response to glucose,demonstrating the presence of functional beta cells. Current studies inare focused on further understanding of the variables leading toincreased cell number, insulin content and/or functionality.

Demonstration of a Glucose-stimulated Reversible Calcium Current

A key question to address is how many of the insulin-containing cellsgenerated in culture possess a functional glucose response. One way todetermine this is to measure the number of cells capable of generatingan inward calcium current in response to glucose administration. Insulinsecretion is known to be mediated by an inward calcium current linked toglucose metabolism (Kalkhoff & Siegesmund, 1981, Wang & McDaniel, 1990).To assess the presence and number of functional beta cells within theculture, we measured cytosolic calcium influx in response to glucoseusing the calcium-dependent fluorescent dye Fluo-3. The results of arepresentative experiment (n>10) using NACs is shown in FIG. 8. In thisexample 33% of the sampled cells showed strong amplitude and kinetics ofcalcium current induction in response to elevated glucose. Measurementsof cells still attached to the monolayer failed to detectglucose-induced changes in intracellular calcium.

The amplitude and kinetics of the glucose-induced calcium current thatwe observe in our duct culture-derived beta cells are similar with thosedocumented to occur in islet-derived beta cells (Asada et al. 1998;Schuit, 1996). Cell-to-cell variations in the amplitude and kinetics ofglucose induced calcium currents have been interpreted as evidence ofheterogeneity in beta cell physiology. Single beta cells studied inisolation have been shown to have altered insulin secretion rates andglucose sensitivity in comparison to intact islets (Halban et al. 1982;Bosco et al. 1989), and individual beta cells have been shown to havemarkedly different rates of insulin synthesis (Moitoso de Vargas et al.1997), all of which are observed in our culture. The amplitude variationin calcium currents might also be explained by differences in Fluo-3 dyeloading.

The characteristic calcium current profile was not induced by2-deoxyglucose, a non-metabolized glucose analog (Niki et al. 1974,1993; Malaisse 1979). It was completely inhibited by diazoxide, a highaffinity inhibitor of the SUR-linked potassium channel (Thomas et al.1996) that is necessary for calcium-induced insulin secretion (Henquinet al. 1982; Trube et al. 1986), and was also inhibited by EGTA(Wollheim & Sharp 1981; Wang & McDaniel, 1990), which sequestersextracellular calcium (not shown). It could however be activated bytolbutamide, a high-affinity activator of the SUR-linked potassiumchannel, which is used specifically to stimulate insulin secretion indiabetics (Sato et al. 1999; Melander, 1998). The reversibility of thisstimulated calcium current is demonstrated in FIG. 8B. In theseexperiments (n=3) 10% of the cells could respond reversibly to glucoseand be stimulated finally by the insulin secretagogue tolbutamide.Fifty-five percent of the cells did not respond to either stimulus, andthe remaining 35% of the cells responded either to glucose but nottolbutamide, or to tolbutamide but not glucose, indicating aheterogeneous and complex population. We conclude that, despite havingheterogeneous insulin expression levels, 10-40% of the NACs couldrespond to glucose and thus behave like functional beta cells.

DISCUSSION

We describe here an in vitro culture system that allows the study offunctional beta cell formation from a purified pancreatic ductpopulation. Culture of such ducts resulted both in an increase ininsulin-positive cells over time and an increase in the total number ofduct fragments that became insulin-positive. This latter resultindicates that cells capable of expressing insulin may become activatedduring culture. In addition to these results, we describe the appearanceof an interesting population of non-adherent cells that arises duringculture, and whose numbers and emergence can be directly regulated byaddition of factors and agents.

These non-adherent cells, which we refer to as NACs, are heterogeneousin size, granularity, and marker expression. Immunocytochemical analysisshows that all four islet endocrine markers can be detected within thispopulation. Our analysis also shows that these cells appear in ratiossimilar to their ratios in the adult pancreatic islet, with the insulinexpressing cells being the most numerous, followed by glucagon,somatostatin, and pancreatic polypeptide. Because of their relevance tohuman disease, assessing the number and functionality of theinsulin-expressing cells has been our major focus.

The handpicked duct material contained very few insulin-positive cellsat the start of culture. Analysis of the number of insulin-positivecells at the start and endpoint of culture demonstrated an increase ofup to 500-fold, primarily in the new nonadherent cell population. Sincethe insulin-positive cells observed early in culture rarely incorporatedBrdU, we propose that the majority of the observed beta-like cells arosefrom an expanding precursor within the duct.

The mechanism of DCE stimulation of insulin expression and NAC formationis not known. One of the components, dexamethasone, is a glucocorticoidanalog known to have multiple effects on pancreas, including stimulationof fetal islet differentiation (Korsgren et al. 1993), stimulation ofpancreatic tumor cell growth (Brons et al. 1984), and upregulation ofexocrine marker expression (Rall et al. 1977; Van Nest et al. 1983), butsurprisingly also suppresses insulin expression in mature mouse islets(Lambillotte et al. 1997). Glucocorticoids have also been shown toupregulate EGF receptor expression in some cell types such ashepatocytes (Gladhaug et al. 1989). EGF is an important mitogen andregulator of gastric and pancreatic epithelium (Meittinen 1997), and infact has been shown to stimulate epithelial-like outgrowths in culturedpancreatic ducts (Heimann & Githens 1991). Agents such as cholera toxinwhich raise intracellular cAMP levels have been shown to stimulateepithelial cell properties (Rindler et al. 1979), and cholera toxin incombination with EGF has been shown to induce cyst formation inpancreatic duct and islet cultures (Heimann & Githens, 1991; Yuan et al.1996). Interestingly, Heimann and Githens (1991) used this combinationof DCE to identify and purify ductal epithelium from fibroblasts throughthe stimulation of cyst formation in collagen or agarose-embeddedcultures. In our hands, continuous culture of ducts in DCE also leads tomonolayer formation, but without NAC formation. It may be that thechoices of culture architecture (embedded versus flat monolayer) and thetiming of factor addition are responsible for these differences, andthese are currently being investigated.

Perhaps due to the early and possibly immature nature of the generatedcells, or the fact that the cells have not yet formed the electricalcontacts required for full islet function, the level of hormoneexpression found within individual monolayer or NAC cells was much lowerthan in adult beta cells. Our insulin extraction studies indicated thatthe average NAC cell contains 20-50 fold less insulin than a comparableadult rat beta cell . It may be that only the insulin ebrighti cellspossess a glucose response and that the edimi cells represent lessmature, non-glucose responsive pre-beta cells. Nonetheless, a muchlarger proportion of cells could be demonstrated to possess aglucose-stimulated insulin secretion response in the NAC population thanin a randomly selected population of monolayer cells. It is. likely thatour culture system is missing the trophic influences necessary forstimulating cell-cell contact, full hormone expression and complete betacell maturation. A number of factors have been shown to increase fetalbeta cell insulin expression and enhance insulin secretion (Otonkoski etal., 1993, 1994; Huotari et al., 1998; Sorenson & Brelje, 1997) andthese are currently being tested in our system.

The system we describe herein allows for the first time the in vitrostudy of regenerative and neogenic events that until now have only beendescribed in vivo. We show that the system can be manipulated to give arange of cell identities, that a significant increasie ininsulin-positive cells can be obtained and that within this populationof cells, beta cell-like function can be detected. Our studies indicatethat much of the regenerative and stem cell activities ascribed to thepancreatic duct system by in vivo manipulations can be recapitulatedthrough in vitro culture. This system now makes possible a systematicsearch for those cells responsible for these activities as well as theidentification of factors that influence their numbers, hormone content,and functionality. In addition, this system constitutes the first steptowards achieving the goal of a controlled and defined process to createfunctional beta cells through a naturally occurring, non-cell-engineeredprocess as a therapeutic pathway for the treatment of insulin-dependentdiabetes.

EXAMPLE 2 Induction of Pancreatic Progenitor Cell Differentiation

The monolayer can be grown in the presence of EGF (10 ng/ml) or TGF-a(10 ng/ml) to enhance growth. Induction of differentiation is believedto be cAMP dependent. Agents which induce an increase in intracellularcAMP levels are anticipated to induce differentiation.

The cocktail DCE (1 μM Dexamethasone, 100 ng/ml Cholera toxin, 10 ng/mlEGF induces an increase in the number of insulin positive cells in thecultured duct monolayers. FIG. 9 shows the comparison of monolayerstreated with DCE+5% FCS versus 5% FCS alone. Ducts were cultured forfive days and then treated for an additional 48 hours. Note that thereis an approximate 5-fold increase in the total number of insulinpositive cells in the culture in response to DCE treatment. The totalnumber of cells in the culture also increases by approximately 20%. Thebars represent the average of quadruplicate wells.

Dexamethasone, Cholera toxin, Forskolin, Dibutyrl cAMP and Na-Butyratehave all been tested and found to induce differentiation. FIG. 10 showsthat Forskolin, Dibutyrl cAMP and Na-Butyrate can substitute for DCE ininducing the appearance of floating progenitor cells. Briefly,monolayers of ductal fragments induced after 5 days culture with thecAMP agonists forskolin and dibutyryl cAMP as well as the fetal isletdifferentiating agent sodium butyrate. Both low and high concentrationsof each factor were applied to the duct monolayer. After 48 hours, theresultant NACS were collected and counted. Treatments are shown on thex-axis and number of floating progenitor cells is shown on the y-axis.Each bar is the added total of duplicate wells.

We have also observed that secretin can induce differentiation of themonolayer and the appearance on pancreatic progenitor cells. In FIG. 11,after 5 days of culture, secretin was added to the monolayers in a doserange of 1-100 nM. The number of floating progenitor cells wasdetermined after 48 hours of treatment. Each bar represents the total oftwo combined 1.9 cm² wells.

The duct cultures used in FIG. 12 were cultured as described above.After 48 hours in varying secretin doses, both the number of insulinpositive cells on the monolayer and the total number of floatingprogenitor cells were counted and scored. Scoring of insulin was done byimmunocytochemistry. Note that there is a dose dependent increase in thenumber of floating progenitor cells. The number of insulin positivecells in the monolayer also increases with secretin dose and theapparent decrease in insulin positive cells at the 50 nm dose isanomalous. Each point represents the average of duplicate wells. Theleft hand y-axis denotes the total number of obtained floatingprogenitor cells, and the right hand y-axis denotes the number ofinsulin positive cells per well. Secretin dose is shown on the x-axis.

FIG. 13 demonstrates that Vasoactive Intestinal Peptide (VIP) alsodifferentiates duct monolayers by inducing the appearance of floatingprogenitor cells. After five days in culture, VIP was added to thecultures and the number of induced floating progenitor cells wasdetermined after 48 hours of treatment. C=control and is 5%FCS. Theoptimal dosage in this experiment was 50 ng/ml of VIP, which induceda >3-fold increase int he number of floating progenitor cells versuscontrol. The y-axis denotes the number of such cells (×100). Each bar isthe total of two pooled wells.

We also observed that the presence of insulin diminishessecretin-induced differentiation. See FIG. 14. Floating progenitor cellswere induced with secretin (100 nM). Simultaneous addition of insulin(10 ng/ml) with secretin diminished the overall induction of floatingprogenitor cells. Each bar represents the total of two pooled duplicatewells and the number is expressed on the y-axis as number cells (×100).

EXAMPLE 3 Isolation of Pancreatic Progenitor Cells using LectinCell-surface Marker

We also set out to identify cell-type specific markers which could beused to isolate/purify pancreatic progenitor cells, or thepancreatic/ductal epithelial which gives rise to such progenitor cells.Amongs the various canidate reagents we tested, we discovered thatcertain lectins preferentially bound to, and therefor facilitate theisolation of, duct epithelial cells ultimately able to producepancreatic progenitor cells.

Arachis hypogaea (Peanut Agglutinin, PNA) is a plant lectin that bindsto specific carbohydrate groups on cell surfaces. PNA binds togalactosyl (β1,3) N-acetyl galactosamine. It was initially selected forstudy as a beta cell marker (ref: Heald K A, Hail C A, Hurst R P, KaneN, Downing R, Diabetes Res 1991 May;17(1):1-6, Separation of beta-cellsfrom dispersed porcine pancreas by selective lectin binding); however,in our hands, PNA did NOT label islet cells from rat, but DID label ductepithelial cells.

PNA (Arachis hypogaea, Peanut Agglutinin) was obtained from VectorLaboratories., FITC-conjugated (Cat.# FL-1071) and used at 1:250-500dilution.

Paraffin sections of adult human pancreas were obtained from CarolinaBiological Supply.

Protocol for Histochemistry:

Paraffin sections of adult human pancreas, adult rat pancreas, embryonicrat pancreas, or cryosections of adult mouse pancreas were used.Alternatively, Time 0 duct or cultured duct preparations from 2 week oldrat pancreas were examined, with or without paraformaldehyde fixation.PNA-FITC was used usually at 1:250 dilution in PBS or DMEM/HEPES mediumand incubated for 1 hr to overnight at 4° C., then washed and mountedunder VectaShield mounting medium containing DAPI. Cells stained withoutprior fixation were post-fixed before addition of mounting medium.

Protocol for FACS:

PNA-FITC was diluted 1:250 in sterile wash buffer (Ca++ Mg++-free PBScontaining 1% FBS). Dispersed live cells (approx. 2×106 cells) were spundown and resuspended in 100 μl of lectin and incubated for 30-45 min at4° C. Cells were then washed twice with sterile wash buffer, resuspendedin 2 ml of Iscoveis modified DMEM containing 5% FBS, Pen/Strep, 1 mMglutamine, and held on ice until run on the FACSVantage. Standard FACSprocedures were used.

FACS sorted cells were collected into tubes or delivered directly intomultiwell culture plates containing complete Iscoveis medium (seeabove). Cell density at seeding, surface, substrates, and culture timeswere varied. Some cultures were re-analyzed by FACS, and some wereanalyzed by histochemistry.

2 week rat duct, Percoll prep ↓ Dissociation to single cell ↓ Incubationof live single cells with PNA-FITC ↓ FACS and recovery of PNA+ and PNA−cells ↓ Culture of sorted cells (currently 2 weeks) ↓ Analysis fordifferentiated cell types: Histochemistry, LDL uptake, etc. ↓ Testing offactors that cause (e.g.,) proliferation, retention of PNA+ character,or differentiation to islet subtypes ↓ In vitro growth &/ordifferentiation ↓ Expansion, implantation into animals for rescue

Using PNA, we made the following observations:

(i) PNA as a marker for Epithelial Cells. PNA marks the single layer ofepithelial cells in the pancreatic duct. It does NOT mark islet cells inrat, in contrast to the report in the literature of beta cell marking inporcine islets. It does not mark blood vessels or stromal cells, byimmunohistochemistry. PNA marks the epithelial cells of pancreatic ductin adult animals as well as the epithelial sheet in embryogenesis (shownat stages e15, e16 and e18 in rat).

(ii) PNA labels cell surfaces.

(iii) PNA labels live, unfixed, unpermeabilized cells.

(iv) PNA does not mark the major pancreatic duct (common bile duct,CBD). PNA marks primarily the medium-sized interlobular ducts and manyof the larger intralobular ducts.

(v) PNA is a suitable reagent for Fluorescence Activated Cell Sorting.PNA allows a viable cell sorting and recovery by FACS (Becton DickinsonFACSVantage); PNA-positive cells can be sorted directly into multiwellplates. We have applied PNA labeling to RIN, islet cells, T0 duct, andcultured duct monolayers. Approximately 5-15% of a T0 duct prep isPNA-positive by FACS analysis. The percentage does not seem to changevery much with culture (over 4 days in FBS). PNA-positive sorted cellsare 76-94+% pure upon reanalysis, depending on the selectivity of thesort (events per drop, sort selection mode, etc.). The PNA-negativepopulation is 99+% negative.

(vi) PNA-sorted cells have favorable Growth Characteristics. Cells areviable but nonadherent after sorting. Dispersed duct cells take aminimum of 7 days to adhere to substrate and begin to grow. In contrast,whole single ducts sit and begin to spread after 24 hr. Cell viabilityis dependent on plating density in the absence of culture additives. Anunsorted population of cells ({grave over (1)}PreSort{circumflex over(1)}) proliferates readily; PNA-positive sorts are slowest. Theimplication is that other cell types besides the PNA-positive cells arerequired for maintaining healthy outgrowth, as well as certain cellcharacteristics (below). PNA-positive cells in culture do not remainPNA-positive (cells restained with PNA-FITC).

A distinct population of cuboidal endothelial-like cells is prominent;also flatter, larger, more fibroblastic cells are present. The formerexhibit uptake of DiI-conjugated acetylated-LDL, a characteristic ofendothelial cells, while the larger flatter cells do not.

The unsorted and PNA-negative populations grow into cultures of verymixed phenotype and morphologies. A very small percentage of cells inthese cultures are diI-Ac-LDL-positive (i.e., endothelial-like).

PNA-positive cells in culture are NOT insulin or PDX-1 positive. Manycells in the mixed PreSort population are positive for both of thesebeta cell markers. By and large, the strong PDX-positive cells are weakor negative for PNA. Strongly PNA-positive cells are not PDX-positive.This suggests a progression from one cell type to another.

A small number of cells in the PNA-negative cell cultures are positivefor Insulin and PDX-1. This suggests either that a small number ofPDX+cells (that were PNA-negative) were recovered, or that the presenceof the other cell types has activated PDX-1 expression, perhaps from asmall carryover of PNA-positive progenitor cells.

PDX-1 expression is much higher (more numerous and relatively brighter)than Insulin expression in these cultured duct preps. Since PDX-1protein is a regulator of Insulin expression, this finding also suggestsa progression, from PDX-1 positive to Insulin-positive.

Glucagon-positive cells outnumber PYY-positive cells, although both arerare in all sort fractions. Previous work has indicated thatPYY-positive cells precede appearance of Glucagon cells; thus theseresults would suggest progenitor cells have already progressed beyondthis point.

Based on these findings, we conlude that the ability of PNA toselectively detect pancreatic duct epithelial cells may permit therecovery of a population of cells containing an islet progenitor celltype. These cells in themselves appear to be insufficient to survive anddifferentiate; that is, other cells or factors may potentiateproliferation and differentiation. Nonetheless, PNA-selection representsa large step forward in being able to perform recombination experimentsto identify the components necessary to grow pancreatic islets.

FIGS. 15-17 illustate the phenotype of cells which have been culturedfor two weeks after being sorted on the basis of PNA staining.

FIGS. 18 and 19 illustrate the specificity of PNA in adult and embryonicpancreas.

FIGS. 30 and 31 illustrate the binding of other lectins to adult ratpancreas.

FIGS. 32-39 illustrate the specificity of binding of lectins to adulthuman pancreas.

EXAMPLE 4 Identification of Genotype of Pancreatic Progenitor Cells

In order to improve our technique for isolating pancreatic progenitorcells, we have designed a protocol for determining the identity of apancreatic beta cell, or it precursor, in terms of its gene expressionprofile. In general, the method applies single cell cDNA amplificationto gene expression analysis. In such a manner, the gene expression“fingerprint” for a cell at a particular stage of development can beobtained by arrayed hybridization.

Briefly, single cells are isolated, e.g., from pancreatic tissue, ThecDNA from each cell are amplified by the single cell PCR developed byBrail et al. (1999) Mutat Res 406: 45-54, and labelled with P³². ThecDNAs are then selected for existence of particular messages, e.g.,insulin and PDX1.

CDNAs of known pancreatic markers are generated by PCR and arrayed onnylon membranes. The resulting assays are used to hybridize with thelabelled single cell cDNAs. The autoradiograph images of the array canbe used to define and identify the gene expression profile for anindividual cell.

FIGS. 20-29 further illustate the protocol. FIG. 20 shows the results oftypical single cell mRNA PCR amplification reactions. FIG. 21illustrates the changes in the gene expression during pancreaticdevelopment, as determined by the subject method. FIG. 22 illustratesone embodiment of an array of markers for detecting beta cells andprecursors thereof.

FIG. 23 shows typical autoradiographs profiling gene expression in adultand embryonic pancreatic tissue, and heart. FIG. 24 demonstates howquantatitve analysis of gene expression can be carried out as part of adetermination of the gene expression profile of a cell. Likewise, FIG.25 shows autoradiographs profiling gene expression in embryonicpancreatic tissue at different stages and after different stimulus; FIG.26 illustate the quantatitve analysis of the autoradiagraphs.

FIG. 27 shows autoradiographs profiling gene expression in the so-calledfloating progenitor cells described in the examples above; and FIG. 28illustate the quantatitve analysis of the autoradiagraphs of FIG. 27.Using the subject method, we have demonstrated that our pancreaticprogenitor cells are PNA⁺ and PDX1⁻ when they are first isolated. As thecells differentiate to insulin-secreting cells (insulin⁺), they becomePNA⁺, PDX1⁺. The earlier progenitor cells, in addition to being PNA⁺,PDX1⁻, insulin⁻, PYY⁻, glucagon⁻ and cytokeratin⁺.

FIG. 29 shows the relative levels of expression of certain genes betweenadult islets and during pancreatis development.

EXAMPLE 5 Implanted Cells from a Pancreatic Duct-derived CultureTransiently Rescues the Diabetic State

SCID/icrl mice were obtained from Taconic. Average weight per mouse was25 g. Streptozotocin (STZ) was purchased from Sigma and made into a 30mg/ml solution in 20 mM sodium citrate buffer, pH 4.5. Each animalreceived STZ at a dose of 200 mg/kg and fed ad libitum prior to fastingblood glucose (food removed the previous evening) on the morning of the3^(rd) day post injection. Diabetic animals were those found to haveblood glucose in excess of 200 mg/dl with the average centering around300 mg/dl. Insulin pellets (Innovative Research of America) thatreleased 1.2 U of porcine insulin per day for 7 days were then implantedvia trochar subcutaneously over the scapula. After monitoring forrecovery from the diabetic state (blood glucose=100 mg/dl), either cellsderived from pancreatic duct culture, or isolated adult pancreaticislets were then implanted under the renal capsule using standardsurgical procedures. Either 500,000 or 10⁶ cells (non-adherent cell(NAC) fraction of duct culture) were implanted into mice. All operatedmice survived in placebo, islet, and insulin pellet only groups. 6/7cell implanted mice died 48-72 hours post implantation. For all groupsfasting blood glucose was measured at standard intervals by tail bleed.

As shown in FIG. 40, a heterogeneous population containing functionalbeta cells derived from the non-adherent portion of a differentiatedpancreatic duct monolayer was implanted into streptozotocin(STZ)-treated diabetic mice. SCID mice injected with STZ became diabeticwithin 48 hours. Insulin containing pellets were then implantedsubcutaneously to stabilize the blood glucose and create a more stableenvironment for cell implantation. The insulin pellet was designed toexpire 7 days post-implantation at T=11 days (T11). Within 48 hours ofpellet implant the fasting blood glucose of these animals were reducedfrom a range of 280-380 mg/dl blood glucose to less than 50 mg/dl. Intest groups either cells or adult islets as positive control were thenimplanted under the renal capsule. One week later (T13) fasting bloodglucose was measured and again at days 16, 21, and 28. Black squaresrepresent placebo group (n=5 mice) and as expected, in the absence ofinsulin, the blood glucose slowly climbed over time to well over 300mg/dl. Animals (n=5) implanted with insulin pellets only and no cellimplants also performed as expected, with a transient rescue followed bydiabetic rebound after the insulin release tablet had expired (reddiamonds). Animals receiving islets (blue triangles, n=5, 400 islets peranimal) showed perfect long term rescue with fasting blood glucose beingmaintained at approximately 100 mg/dl. The single surviving animalreceiving duct-derived cells (green circles, n=1 of 7) showed atransient rescue of the diabetic state. The single animal demonstrated a4-5 day lowering of >150 mg/dl blood glucose before rebounding topre-implant blood glucose levels.

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All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 1 Immunostaining in Duct Cultures Marker expression in isolatedducts and Insulin in duct cultures. Marker Expression in PancreaticDucts at the Start of Culture Insulin PDX-1 PYY Amylase % Positive Ducts 7.8%  5.3%  8.3%% 14.5% % Positive Cells 0.01% 0.01% 0.015% 0.02%Immunostaining for Insulin in Duct Cultures Total Ins Total CellsPositive % Insulin- Prep Cells/Prep Positive T = 0, Primary Ducts   800K   80 0.01% T = 7 days Control Monolayer 2,000K   400 0.02% (5x ↑)Activated (+ DCE) Monolayer 2,400K   1000 0.04% (12.5x ↑)   NACs   100K40,000   40% (500x ↑)  The average cell number per duct fragment was3450 ± 1860 (n = 10), comprising a single layer of epithelial cellssurrounded by mesenchymal/stromal cells. Positive immunostaining onwhole isolated ducts (n = 60-90) was as follows: Insulin 7/90 ducts, 32positive cells; PDX-1 4/75 ducts, 15 positive cells; PYY 5/60 ducts, 31cells; Amylase 10/69 ducts, 49 cells. n = 2 isolations for PDX-1, PYY,amylase; n = 4 isolations for insulin. NAC yield generally ranged from80,000 to 130,000 cells/prep; maximum seen was 160,000 althoughtheoretical maximum based on an observed cell density is estimated to be300,000 cells/prep (10,000 NACs/cm²).

TABLE 2 Immunostaining for PDX-1, PYY and Amylase in Duct Cultures TotalPDX-1 % PDX-1 Positive Cells/Prep Positive T = 0, Primary Ducts  80 0.01% T = 7 days Control Monolayer  480  0.02% (6x ↑) Activated (+ DCE)Monolayer 1200  0.05% (15x ↑)  Total PYY % PYY Positive Cells/PrepPositive T = 0, Primary Ducts  120 0.015% T = 7 days Control Monolayer 60 0.003% (2x ↓) Activated (+DCE) Monolayer  100 0.004% (approx. nochange) Total Amylase % Amylase Positive Cells/Prep Positive T = 0,Primary Ducts  160  0.02% T = 7 days Control Monolayer * ** (>10x ↓)   Activated (+ DCE) Monolayer ND ND 96-100% of Insulin-positive cells inculture were also PDX-1-positive. In addition, PDX-1-positive,insulin-negative cells appear in all states of culture; however, inDCE-activated monolayers, the number of these cells is increased (20-50%more cells) and there are more cytoplasmic PDX-1 cells. There is littleor no apparent change in PYY in the monolayer; however, PYY appears tobe present in emerging NACs. *Only two out of ten preparations containedany Amylase-positive cells at all. **% Amylase-positive is on the orderof 0.001% for the positive preps. ND, not detected.

9 1 60 DNA Artificial Sequence Description of Artificial Sequence primer1 atgtcgtcca ggccgctctg gacaaaatat gaattctttt tttttttttt tttttttttt 60 221 DNA Artificial Sequence Description of Artificial Sequence primer 2cacaactgga gctgggtgga g 21 3 24 DNA Artificial Sequence Description ofArtificial Sequence primer 3 caaaggcttt attcattgca gagg 24 4 20 DNAArtificial Sequence Description of Artificial Sequence primer 4gaccgcaggc tgagggtgag 20 5 21 DNA Artificial Sequence Description ofArtificial Sequence primer 5 cagaggtctg ccagcatctc g 21 6 21 DNAArtificial Sequence Description of Artificial Sequence primer 6tcccagaaga agtcgccatt g 21 7 22 DNA Artificial Sequence Description ofArtificial Sequence primer 7 ttcattccgc agagatgttg tg 22 8 22 DNAArtificial Sequence Description of Artificial Sequence primer 8aagtccctca ccctcccaaa ag 22 9 22 DNA Artificial Sequence Description ofArtificial Sequence primer 9 aacacctcaa accactccca gg 22

What is claimed is:
 1. A method of isolating progenitor cellscomprising: i) obtaining pancreatic ductal tissue substantially free oftissues other than the ductal epithelium and surrounding mesenchyme, ii)obtaining dissociated pancreatic ductal cells from the pancreatic ductaltissue; iii) culturing said pancreatic ductal cells in a suitablenutrient medium; and iv) isolating a population of progenitor cells fromsaid culture.
 2. A method of claim 1, wherein obtaining pancreaticductal cells comprises obtaining pancreatic intralobular ductalepithelial cells.
 3. A method of claim 1, wherein said pancreatic ductalcells are obtained by explant or by enzymatic digestion.
 4. A method ofclaim 1, wherein culturing said pancreatic ductal cells comprisesgrowing said cells to confluence.
 5. A method of claim 1, wherein saidprogenitor cells are isolated by mechanical separation.
 6. A method forpreparing a composition comprising progenitor cells, comprising i)isolating pancreatic progenitor cells by the method of claim 1, and ii)combining the pancreatic progenitor cells with a pharmaceuticallyacceptable excipient.
 7. The method of claim 1, wherein the pancreaticducts are substantially free of islet tissue.
 8. A method of claim 4,wherein isolating a population of progenitor cells comprises isolatingnon-adherent cells, and wherein the method further comprises treatingthe isolated cells with an agent.
 9. The method of claim 6, whichprogenitor cells are mammalian.
 10. The method of claim 6, whichprogenitor cells differentiate to pancreatic lineages.
 11. The method ofclaim 6, which progenitor cells can be maintained in culture for atleast about 7 days.
 12. The method of claim 6, which progenitor cellsare inducible to differentiate into pancreatic islet cells.
 13. Themethod of claim 6, further comprising combining the cells and/or theexcipient with an agent that induces differentiation of the progenitorcells.
 14. A method of claim 8, wherein said agent inducesdifferentiation and is selected from forskolin, dibutyryl cAMP,Na-butyrate, dexametbasone, and cholera toxin.
 15. A method of claim 8,wherein said agent is a growth factor.
 16. The method of claim 9,wherein the progenitor cells are transgenic mammalian cells.
 17. Themethod of claim 9, wherein the progenitor cells are primate cells. 18.The method of claim 9, wherein the progenitor cells are miniature swinecells.
 19. The method of claim 10, which progenitor cells differentiateto β islet cells, α cells, δ islet cells, φ islet cells, or exocrinecells.
 20. The method of claim 12, which islet cells are pancreatic βislet cells.
 21. The method of claim 12, which islet cells arepancreatic α islet cells.
 22. The method of claim 12, which islet cellsare pancreatic δ islet cells.
 23. The method of claim 12, which isletcells are pancreatic φ islet cells.
 24. The method of claim 13, whereinsaid agent is selected from forskolin, dibutyryl cAMP, Na-butyrate,dexamethasone, and cholera toxin.
 25. A method of claim 15, wherein saidgrowth factor is selected from insulinlike growth factor (IGF),transforming growth factor (TGF), fibroblast growth factor (EGF),epidermal growth factor (EGF), hepatocyte growth factor (HGP), hedgehog,and vascular endothelial growth factor (VEGF).
 26. A method of claim 15,wherein said growth factor is selected from the transforming growthfactor-β (TGFβ) superfamily, bone morphogenic protein-2 (BMP2) and bonemorphogenic protein-7 (BMP7).
 27. The method of claim 17, wherein theprogenitor cells are human cells.
 28. A method for stimulating the exvivo proliferation of mammalian pancreatic β-islet cells, comprising thesteps of: (a) preparing a primary culture of mammalian pancreatic cells;and (b) contacting said primary culture cells with a cAMP agonist in anamount sufficient to induce the primary culture to differentiate toβ-islet cells.
 29. The method of claim 28, wherein the mammalianpancreatic cells are human pancreatic cells.
 30. The method of claim 28,wherein inducing the primary culture to differentiate comprises inducingan increase in average cellular insulin production.
 31. The method ofclaim 28, further comprising growing said primary culture cells in amonolayer on an extracellular matrix in the presence of a growth factor.32. The method of claim 28, further comprising contacting said primaryculture cells or β-islet cells with an agent that upregulates theinsulin gene.
 33. A method for preparing a composition comprisingβ-islet cells, comprising i) isolating β-islet cells by the method ofclaim 28, and ii) combining the β-islet cells with a pharmaceuticallyacceptable excipient.
 34. The method of claim 32, wherein the agent is apoly (ADP-ribose) synthetase inhibitor.
 35. The method of claim 34,wherein the poly(ADP-ribose)synthetase inhibitor is nicotinamide or abenzamide.
 36. A method for stimulating the ex vivo proliferation ofhuman adult pancreatic beta islet-cells, comprising the steps of: (a)preparing a monolayer culture of primary human adult pancreatic cells;and (b) culturing said cells with a cAMP agonist in an amount sufficientto induce the primary culture to produce insulin-producing cells.
 37. Amethod for preparing a composition comprising insulin-producing cells,comprising i) producing insulin-producing cells by the method of claim36, and ii) combining the insulin-producing cells with apharmaceutically acceptable excipient.
 38. A method of producing humanadult pancreatic islet cells in clinically useful quantities,comprising: (a) seeding a bioreactor with a human pancreatic cellculture; (b) perfusing said bioreactor with a complete growth mediumsupplemented with an amount of a cAMP agonist sufficient to induce cellsin the bioreactor to proliferate and differentiate intoinsulin-secreting cells; and (c) harvesting insulin-secreting cells fromsaid bioreactor.
 39. A method for preparing a composition comprisinginsulin-secreting cells, comprising i) producing insulin-secreting cellsby the method of claim 38, and ii) combining the insulin-secreting cellswith a pharmaceutically acceptable excipient.